PYK2 and inflammation

ABSTRACT

The present invention relates generally to the fields of immunology and medicine, and more specifically to the field of cellular signal transduction. The present invention relates, inter alia, to methods for diagnosis, treatment, and identification of therapeutics for particular inflammation-related diseases or disorders characterized by an interaction between a PYK2 polypeptide and a natural binding partner.

RELATED APPLICATIONS

[0001] The present application claims priority to U.S. ProvisionalPatent Application Ser. No. 60/114,465 by Schlessinger, Okigaki, andGishizky, entitled PYK2 and Inflammation, filed Dec. 30, 1998 (Lyon &Lyon Docket No. 236/075) which is hereby incorporated by referenceherein in its entirety, including any drawings, tables, or figures. Inaddition, the present application is related to U.S. application Ser.No. 08/357,642, by Lev and Schlessinger, Lyon & Lyon Docket No. 209/070,entitled “PYK2 related Products and Methods”, filed Dec. 15, 1994; Ser.No. 08/460,626, by Lev and Schlessinger, Lyon & Lyon Docket No. 211/121,entitled “PYK2 related Products and Methods”, filed Jun. 2, 1995; andSer. No. 08/987,689 by Lev and Schlessinger, Lyon & Lyon Docket No.230/110, entitled “PYK2 related Products and Methods”, filed Dec. 9,1997, all of which are hereby incorporated by reference herein in theirentirety including any drawings, figures, or tables.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the fields ofimmunology and medicine, and more specifically to the field of cellularsignal transduction.

BACKGROUND OF THE INVENTION

[0003] None of the following discussion of the background of theinvention, which is provided solely to aid the reader in understandingthe invention, is admitted to be or to describe prior art to theinvention.

[0004] Cellular signal transduction is a fundamental mechanism wherebyexternal stimuli that regulate diverse cellular processes are relayed tothe interior of cells. One of the key biochemical mechanisms of signaltransduction involves the reversible phosphorylation of tyrosineresidues on proteins. The phosphorylation state of a protein is modifiedthrough the reciprocal actions of tyrosine phosphatases (TPs) andtyrosine kinases (TKs), including receptor tyrosine kinases andnon-receptor tyrosine kinases.

[0005] RTKs are composed of at least three domains: an extracellularligand binding domain, a transmembrane domain and a cytoplasmiccatalytic domain that can phosphorylate tyrosine residues. Theintracellular, cytoplasmic, non-receptor protein tyrosine kinases do notcontain a hydrophobic transmembrane domain or an extracellular domainand instead contain non-catalytic domains in addition to their catalytickinase domains. Such non-catalytic domains include the SH2 domains andSH3 domains. The non-catalytic domains are thought to be important inthe regulation of protein-protein interactions during signaltransduction.

[0006] FAK (focal adhesion kinase) and PYK2 (proline-rich tyrosinekinase also known as RAFTK, CAK and CADTK (Lev, et al. (1995) Nature376:737-745; Avraham, et al. (1995) J. Biol. Chem. 270:27742-27751;Sasaki, et al. (1995) J. Biol. Chem. 270:21206-21219; Yu, et al. (1996)J. Biol. Chem. 271:29993-29998) comprise one family of protein tyrosinekinases. FAK and PYK2 exhibit approximately 45% amino acid sequenceidentity to each other and each contain an N terminus with similarity toband 4.1 homology domain, a centrally located protein tyrosine kinasedomain (Girault, et al. (1999) Trends Neurosci. 22:257-263), and twoproline rich regions at the C-terminus (Lev, et al. (1995) Nature376:737-745). PYK2 and FAK bind to proteins that have been shown tointeract with the cytoskeleton such as paxillin (Salgia, et al. (1996)J. Biol. Chem. 271:31222-31226), p130^(cas), the rhoGAP protein Graf(Ohba, et al. (1998) Biochem. J. 330:1249-1254) and a novel proteincontaining a LIM domain (Matsuya, et al. (1998) J. Biol. Chem.273:1003-1014; Lipsky, et al. (1998) J. Biol. Chem. 273:11709-11713).

[0007] PYK-2 is a non-receptor tyrosine kinase that is activated bybinding of ligand to G-coupled protein receptors such as bradykinin andacetylcholine. PYK2 has a predicted molecular weight of 111 kD andcontains five domains: (1) a relatively long N-terminal domain; (2) akinase catalytic domain; (3) a proline rich domain; (4) another prolinerich domain; and (5) a C-terminal domain.

[0008] PYK2 is expressed in various tissues, including neural tissues,hematopoietic cells, and in some tumor cell lines. PYK2 is believed toregulate the activity of potassium channels in response toneurotransmitter signaling. PYK2 may also regulate ion-channel functionby tyrosine phosphorylation.

[0009] PYK2 is activated by stimulation of G-protein coupled receptors(Lev, et al. (1995) Nature 376:737-745), by stimulation of antigenreceptors on T cells (Qian, et al. (1997) J. Exp. Med. 185:1253-1259); Bcells (Astier, et al. (1997) J. Biol. Chem. 272:228-232), and mast cells(Okazaki, et al. (1997) J. Biol. Chem. 272:32443-32447); as well as inresponse to inflammatory cytokines (Tokiwa, et al. (1996) Science273:792-794; Miyazaki, et al. (1998) Genes Dev. 12:770-775; Takaoka, etal. (1999) EMBO J. 18:2480-2488), and stress signals (Tokiwa, et al.(1996) Science 273:792-794). In some cells, tyrosine phosphorylation andactivation of PYK2 was shown to be triggered by integrin-mediatedadhesion (Astier, et al. (1997) J. Biol. Chem. 272:228-232).Furthermore, PYK2 can be activated by phorbol ester (PMA), or by avariety of extracellular signals that elevate intracellular Ca⁺²concentration (Lev, et al. (1995) Nature 376:737-745).

[0010] It has been proposed that PYK2 acts in concert with Src to linkGi or Gq coupled receptors with the MAP kinase signaling pathway (Dikic,et al (1996) Nature 383:547-550). Autophosphorylation of Y402 on PYK2generates a binding site for the SH2 domain of the docking protein Grb2,and subsequent recruitment of the Grb2/Sos complex leads to activationof the Ras/MAP kinase signal transduction cascade (Dikic, et al. (1996)Nature 383:547-550). Activation of the JNK pathway can also be mediatedby PYK2, and this signaling pathway can be inhibited bydominant-negative forms of rac and cdc42 (Tokiwa, et al. (1996) Science273:792-794). In addition, PYK2 activation leads to suppression ofoutward potassium currents via tyrosine phosphorylation of the delayedrectifier-type potassium channel Kv1.2 (Lev, et al. (1995) Nature376:737-745). PYK2 also interacts with and phosphorylates a family ofphosphatidylinositol transfer proteins, designated Nirs (Lev, et al.(1999) Mol. Cell. Biol. 19:2278-2288), and with a novel ArfGAP,designated Pap (Andreev, et al. (1999) Mol. Cell. Biol. 19:2338-2350),both in vitro and in vivo. In addition, it was shown that activation ofPYK2 leads to tyrosine phosphorylation of other proteins that play arole in signal transmission, including the adaptor proteins Shc (Lev, etal. (1995) Nature 376:737-745) and Cas (Astier, et al. (1997) J. Biol.Chem. 272:228-232).

[0011] PYK2 is activated by extracellular signals that lead to calciuminflux or calcium release from internal stores. PYK2 is phosphorylatedon tyrosine residues in response to a variety of external stimuli. PYK2may provide a link between G-protein coupled receptors and calciuminflux and the MAP kinase signaling pathway, a pathway that relayssignals from the cell surface to regulate transcriptional events in thenucleus.

[0012] In the PCT Publication WO 98/07870 (Avraham, et al.), the authorsdiscuss PYK2 and state that

[0013] “ . . . RAFTK therapeutics which modulate RAFTK activity in Bcells, T cells, and monocytes can be used to treat immune-mediateddisorders and mediate both cell mediated and humoral immune responses.

[0014] Normal hematopoietic cells are dependent on growth factors forgrowth and differentiation and the loss of this growth factor dependencecan lead to autonomous growth. The involvement of RAFTK in severalgrowth factor signaling pathways indicates that misse[x]pression ofRAFTK can lead to the development of cancers, and the present inventioncontemplates modulating RAFTK expression and/or activity to controlaberrant cell growth. In a preferred embodiment RAFTK is modulated totreat cancers of hematopoietic cells. In another embodiment malignancycan be suppressed in certain cells e.g., leukemic cells, by modulatingRAFTK to induce cellular differentiation . . . .

[0015] . . . In one embodiment the RAFTK proteins of the presentinvention can modulate the differentiation or maturation ofhematopoietic cells; the subject RAFTK polypeptides are capable ofinfluencing both the differentiation and maturation of pluripotent stemcells and the proliferation of differentiated cells.”

SUMMARY OF THE INVENTION

[0016] The present invention relates, inter alia, to methods foridentification of compounds useful to treat or preventinflammation-related diseases or disorders characterized by aninteraction between a PYK2 polypeptide and a natural binding partner. Inaddition, the present invention relates to methods for diagnosis and fortreatment of inflammation-related diseases or disorders characterized byan interaction between a PYK2 polypeptide and a natural binding partner.

[0017] The current invention demonstrates for the first time in an invivo mouse model and a cellular model the link between PYK2 and theinflammatory response. To demonstrate the role of PYK2 in vivo, knockoutmice lacking the pyk2 gene were created using molecular genetictechniques. The inflammatory response of the knockout mice was comparedwith the corresponding mice not containing a pyk2 deletion. Experimentsare described in detail herein in the Detailed Description of theInvention.

[0018] The data from these experiments confirm the role for PYK2 incytokine release and support the importance of PYK2 function ininflammation. These experiments indicate that treatments that inhibitthe functioning of PYK2 will be useful to decrease excessiveinflammatory responses, whereas treatments to enhance the functioning ofPYK2 will be useful to augment inadequate immune responses.

[0019] In a first aspect, the invention features a method foridentifying one or more potential compounds useful to treat or toprevent a disease or disorder, wherein said disease or disorder ischaracterized by a inflammatory response, wherein said inflammatoryresponse is characterized by an abnormality in a signal transductionpathway, and wherein said signal transduction pathway includes aninteraction between a PYK2 polypeptide and a natural binding partner,comprising assaying said one or more potential compounds for those ableto modulate said interaction as an indication of a useful said compound.

[0020] By “identifying” is meant investigating for the presence orabsence of a property. The process may include measuring or detectingvarious properties, including the level of signal transduction and thelevel of interaction between a PYK2 polypeptide and a natural bindingpartner.

[0021] The term “compound” preferably refers to a non-peptide organicmolecule, and most preferably refers to a non-peptide synthetic organicmolecule. The term “non-peptide molecule” refers to a compound that isnot a polymer of amino acids. A non-peptide molecule preferably does notcontain chemical moieties that hydrolyze in physiological conditions,e.g. a peptidomimetic. Alternatively, a non-peptide molecule may be apeptoid, or modified nucleic acid molecule. Examples of compounds areincluded in the Description of the Invention, herein. Preferably, suchmolecules have a molecular weight less than 3,000.

[0022] By “inflammatory response” is meant a protective responseelicited by injury or destruction of tissues, which serves to destroy,dilute, or wall off (sequester) both the injurious agent and the injuredtissue. Histologically, it involves a complex series of events,including dilatation of arterioles, capillaries, and venules, withincreased permeability and blood flow; exudation of fluids, includingplasma proteins; and leukocyte migration into the inflammatory focus. Apathologic inflammatory response may be a continuation of an acute formor a prolonged low-grade form, and usually causes permanent tissuedamage. Macrophage and T-cell recruitment and functions, such ascytokine production, directly contribute to inflammatory pathogenesis.There are many types of diseases and disorders associated withinflammatory responses, all of which are intended to be included underspecific embodiments of the present invention.

[0023] An “organism” can be single or multi-cellular. The term includesmammals, and, most preferably, humans. Preferred organisms include mice,as the ability to treat or diagnose mice is often predictive of theability to function in other organisms such as humans.

[0024] By “disease or disorder” is meant a state in an organism (e.g., ahuman) which is recognized as abnormal by members of the medicalcommunity. The disease or disorder is characterized by an abnormality inone or more signal transduction pathways in a cell, where the componentsof the signal transduction pathway include a PYK2 polypeptide and anatural binding partner.

[0025] By “abnormality” is meant a level which is statisticallydifferent from the level observed in organisms not suffering from such adisease or disorder and may be characterized as either an excess amount,intensity or duration of signal or a deficient amount, intensity orduration of signal. The abnormality in signal transduction may berealized as an abnormality in cell function, viability ordifferentiation state. Such abnormality in a pathway can be alleviatedby action at the PYK2:natural binding partner interaction site in thepathway. An abnormal interaction level may also either be greater orless than the normal level and may impair the normal performance orfunction of the organism. Thus, it is also possible to screen for agentsthat will be useful for treating a disease or condition, characterizedby an abnormality in the signal transduction pathway, by testingcompounds for their ability to affect the interaction between a PYK2polypeptide and a natural binding partner, since the complex formed bysuch interaction is part of the signal transduction pathway. However, insome embodiments, the disease or condition may be characterized by anabnormality in the signal transduction pathway even if the level ofinteraction between the PYK2 polypeptide and natural binding is normal.Further in some embodiments, the defect may result from an inability ofa natural binding partner to perform a function on PYK2, or PYK2 toperform a function on a natural binding partner, or both. Finally, insome preferred embodiments, the abnormality does not fall within what istraditionally meant by the signal transduction pathway, e.g. it mayinvolve an activity of PYK2 that does not directly relate to signaltransduction.

[0026] The term “signal transduction pathway” refers to the moleculesthat propagate an extracellular signal through the cell membrane tobecome an intracellular signal. This signal can then stimulate acellular response. The polypeptide molecules involved in signaltransduction processes are typically (but not limited to) receptor andnon-receptor protein tyrosine kinases, receptor and non-receptor proteinphosphatases, SRC homology 2 and 3 domains, PDZ domain containingproteins, phosphotyrosine binding proteins (SRC homology 2 (SH2) andphosphotyrosine binding (PTB and PH) domain containing proteins), PTB(phosphotyrosine binding) domain that binds to phosphotyrosine as wellas non-phosphorylated peptides, PH (pleckstrin homology) domain thatbinds to phosphoinositides, proline-rich binding proteins (SH3 domaincontaining proteins), nucleotide exchange factors, and transcriptionfactors.

[0027] By “interact” is meant any physical association betweenpolypeptides, whether covalent or non-covalent. This linkage can includemany chemical mechanisms, for instance covalent binding, affinitybinding, intercalation, coordinate binding and complexation. Examples ofnon-covalent bonds include electrostatic bonds, hydrogen bonds, and Vander Waals bonds. Furthermore, the interactions between polypeptides mayeither be direct or indirect. Thus, the association between two givenpolypeptides may be achieved with an intermediary agent, or several suchagents, that connects the two proteins of interest (e.g., a PYK2polypeptide and a natural binding partner). Another example of anindirect interaction is the independent production, stimulation, orinhibition of both a PYK2 polypeptide and natural binding partner by aregulatory agent. Depending upon the type of interaction present,various methods may be used to measure the level of interaction. Forexample, the strengths of covalent bonds are often measured in terms ofthe energy required to break a certain number of bonds (i.e., kcal/mol).Non-covalent interactions are often described as above, and also interms of the distance between the interacting molecules. Indirectinteractions may be described in a number of ways, including the numberof intermediary agents involved, or the degree of control exercised overthe PYK2 polypeptide relative to the control exercised over the naturalbinding partner.

[0028] By “a PYK2 polypeptide” is meant the PYK2 polypeptide describedin U.S. Pat. No. 5,837,815 to Lev et al. and WO publication WO 96/18738by Lev et al., both hereby incorporated by reference herein in theirentirety including tables, figures, and drawings. The isolation andcharacterization of the PYK2 polypeptide is also fully describedtherein. The PYK2 polypeptide can be encoded by a full-length nucleicacid sequence or any portion of the full-length nucleic acid sequence,so long as a functional activity of the polypeptide is retained.Preferred functional activities include, but are not limited to, theability to phosphorylate and regulate RAK and/or other potassiumchannels. A variety of methodologies known in the art can be utilized toobtain PYK2 polypeptides for use in the methods of the invention.

[0029] The term “natural binding partner” refers to polypeptides,lipids, small molecules, or nucleic acids that bind to kinases in cells.A change in the interaction between a kinase and a natural bindingpartner can manifest itself as an increased or decreased probabilitythat the interaction forms, or an increased or decreased concentrationof kinase/natural binding partner complex. Binding is understood toinclude interactions such as phosphorylation or dephosphorylation, forexample.

[0030] The term “modulates” refers to the ability of a compound to alterthe interaction of PYK2 and a natural binding partner. The K_(m) of acompound is preferably between 100 μM and 1 μM, more preferably between1 μM and 100 nM, most preferably between 100 nM and 1 nM. A modulatorpreferably promotes or disrupts the interaction of PYK2 and a naturalbinding partner. Alternatively, the modulator may increase or decreasethe cellular activity of the kinase, including phosphorylation. Kinaseactivity is preferably the phosphorylation of a natural binding partneron tyrosine, serine, or threonine residues. Changes in the interactionwith a natural binding partner can also include increasing or decreasingthe probability that a complex forms between the kinase and a naturalbinding partner. A modulator preferably increases the probability thatsuch a complex forms between the kinase and the natural binding partner,and most preferably decreases the probability that a complex formsbetween the kinase and the natural binding partner. In some preferredembodiments the interaction includes actions of the natural bindingpartner on PYK2.

[0031] The term “complex” refers to an assembly of at least twomolecules bound to one another. Signal transduction complexes oftencontain at least two protein molecules bound to one another. Forinstance, a protein tyrosine kinase receptor, GRB2, SOS, RAF, and RASassemble to form a signal transduction complex in response to amitogenic ligand.

[0032] By “disrupt” is meant that the interaction between the PYK2polypeptide and a natural binding partner is reduced either bypreventing expression of the PYK2 polypeptide, or by preventingexpression of the natural binding partner, or by specifically preventinginteraction of the naturally synthesized proteins or by interfering withthe interaction of the proteins.

[0033] By “promote” is meant that the interaction between a PYK2polypeptide and a natural binding partner is increased either byincreasing expression of a PYK2 polypeptide, or by increasing expressionof a natural binding partner, or by decreasing the dephosphorylatingactivity of the corresponding regulatory TP (or other phosphatase actingon other phosphorylated signaling components), by promoting interactionof the PYK2 polypeptide and natural binding partner or by prolonging theduration of the interaction.

[0034] The term “activates” refers to increasing the cellular activityof the kinase. The term “inhibit” refers to decreasing the cellularactivity of the kinase. Kinase activity is preferably thephosphorylation of a natural binding partner on tyrosine, threonine, orserine residues. Changes in the interaction with a natural bindingpartner can also include increasing or decreasing the probability that acomplex forms between the kinase and a natural binding partner. Amodulator preferably increases the probability that such a complex formsbetween the kinase and the natural binding partner, and most preferablydecreases the probability that a complex forms between the kinase andthe natural binding partner.

[0035] In preferred embodiments of methods for screening for compoundspotentially useful for treating or preventing inflammatoryresponse-related diseases or disorders involving the interaction of PYK2and a natural binding partner, the inflammatory response-related diseaseor disorder is selected from the group consisting of inflammatory boweldiseases and connective tissue diseases. Preferably, the inflammatorybowel diseases are selected from the group consisting of ulcerativecolitis and Crohn's Disease and the connective tissue diseases areselected from the group consisting of rheumatoid arthritis, systemiclupus erythematosus, progressive systemic sclerosis, mixed connectivetissue disease, and Sjögren's syndrome.

[0036] Macrophage function and the production of cytokines bymacrophages and other cells associated with the inflammatory responsedirectly contribute to the pathophysiologic progression of the diseases.The importance of PYK2 in these disease processes is indicated by thedecreased production of cytokines in cells from pyk2−/− mice and inmacrophage cell lines expressing kinase inactive PYK2. Thus, inhibitingPYK2 function in inflammatory cells should alleviate some of thepathologic consequences associated with these diseases.

[0037] The term “inflammatory bowel disease” as used herein, refers toinflammatory diseases of the bowel, many of which are of unknownetiology, including Crohn's disease and ulcerative colitis.

[0038] The term “Crohn's Disease” as used herein, refers to a chronicgranulomatous inflammatory disease of unknown etiology, involving anypart of the gastrointestinal tract from mouth to anus, but commonlyinvolving the terminal ileum and/or colon with scarring and thickeningof the bowel wall. It frequently leads to intestinal obstruction andfistula and abscess formation and has a high rate of recurrence aftertreatment.

[0039] By “ulcerative colitis” is meant chronic, recurrent ulceration inthe colon, chiefly of the mucosa and submucosa, of unknown cause. Therectum is almost always involved. It is manifested clinically bycramping abdominal pain, rectal bleeding, and loose discharges of blood,pus, and mucus with scanty fecal particles. Complications includehemorroids, abscesses, fistulas, perforation of the colon, pseudopolyps,and carcinoma.

[0040] The term “connective tissue diseases” as used herein refers toheterogeneous disorders which share certain common features, includinginflammation of skin, joints, and other structures rich in connectivetissue, as well as altered patterns of immunoregulation, includingproduction of autoantibodies and abnormalities of cell-mediatedimmunity. While certain distinct clinical entities may be defined,manifestations may vary considerably from one patient to the next andoverlap of clinical features between and among specific diseases iscommon.

[0041] The term “rheumatoid arthritis” as used herein refers to achronic systemic disease primarily of the joints, usually polyarticular,marked by inflammatory changes in the synovial membranes and articularstructures and by muscle atrophy and rarefaction of the bones.Persistent inflammatory synovitis usually involves the peripheral jointsin a symmetrical fashion, marked by cartilaginous destruction, bonyerosions, and joint deformation. Infiltration of inflammatory cells iscommon. Forms of rheumatoid arthritis include, but are not limited to,juvenile, chronic villous, cricoarytenoid, deformans, degenerative,mutilans, and proliferative.

[0042] The term “systemic lupus erythematosus” as used herein, refers toa disease in which tissues and cells are damaged by deposition ofpathogenic antibodies and immune complexes. B-cell hyperactivity,production of autoantibodies with specificity for nuclear antigenicdeterminants, and abnormalities of T-cell function occur. It may involvevirtually any organ system and follows a course of exacerabationfollowed by remission. A common feature is the alar “butterfly” rash.

[0043] The term “progressive systemic sclerosis” as used herein refersto a multisystem disorder characterized by inflammatory, vascular, andfibrotic changes of skin and various internal organ systems (chiefly GItract, lungs, heart, and kidney). Primary event may be endothelial cellinjury with eventual intimal proliferation, fibrosis, and vesselobliteration. Clinical manifestations include, but are not limited to,Raynaud's phenomenon, scleroderma (fibrosis of the skin), hypertension,and renal failure.

[0044] The term “mixed connective tissue disease” as used herein, refersto syndrome characterized by a combination of clinical features similarto those of systemic lupus erythematosus, progressive systemicsclerosis, polymyositis, and rheumatoid arthritis. Unusually high titersof circulating antibodies to a nuclear ribonucleoprotein are found.Clinical manifestations include Raynaud's phenomenon, polyarthritis andpulmonary fibrosis among others.

[0045] The term “Sjögren's syndrome” as used herein refers to animmunologic disorder characterized by progressive destruction ofexocrine glands leading to mucosal and conjunctival dryness (siccasyndrome). Affected tissues show lymphocyte infiltration andimmune-complex deposition.

[0046] In preferred embodiments of methods of identifying compounds, theone or more compounds modulate (inhibit or promote) the interaction ofPYK2 and a natural binding partner in vitro. An example of an in vitromethod involves growing cells (i.e., in a dish) that either naturally orrecombinantly express a G-coupled protein receptor, PYK2, and RAK. Thetest compound preferably is added at a concentration from 0.1 μM to 100μM and the mixture preferably is incubated from 5 minutes to 2 hours.The ligand is added to the G-coupled protein receptor preferably for 5to 30 minutes and the cells are lysed. RAK is isolated usingimmunoprecipitation or ELISA by binding to a specific monoclonalantibody. The amount of phosphorylation compared to cells that were notexposed to a test compound is measured using an anti-phosphotyrosineantibody (preferably polyclonal). Alternatively, in other methods ofidentifying compounds, the one or more compounds modulate (inhibit orpromote) PYK2 and natural binding partner interactions in vivo.

[0047] In other methods of identifying compounds, the interaction isselected from the group consisting of PYK2 phosphorylation, PYK2 naturalbinding partner phosphorylation, PYK2 de-phosphorylation, PYK2 naturalbinding partner de-phosphorylation, and complex formation between PYK2and a natural binding partner.

[0048] Examples of compounds that could be tested in such screeningmethods include tyrphostins, quinazolines, quinoxolines, quinolines, andindolinones. Publications describing representative examples of thesecompounds and methods of making are given in the Detailed Description ofthe Invention.

[0049] A second aspect of the invention features a method for diagnosisof a disease or disorder, wherein said disease or disorder ischaracterized by an inflammatory response involving an abnormality in asignal transduction pathway that includes an interaction between a PYK2polypeptide and a natural binding partner, comprising detecting thelevel of said interaction as an indication of said disease or disorder.

[0050] By “diagnosis” is meant any method of identifying a symptomnormally associated with a given disease or condition. Thus, an initialdiagnosis may be conclusively established as correct by the use ofadditional confirmatory evidence such as the presence of other symptoms.Current classification of various diseases and conditions is constantlychanging as more is learned about the mechanisms causing the diseases orconditions. Thus, the detection of an important symptom, such as thedetection of an abnormal level of interaction between PYK2 polypeptidesand natural binding partners may form the basis to define and diagnose anewly named disease or condition. For example, conventional cancers areclassified according to the presence of a particular set of symptoms.However, a subset of these symptoms may both be associated with anabnormality in a particular signaling pathway, such as the ras²¹ pathwayand in the future these diseases may be reclassified as ras²¹ pathwaydiseases regardless of the particular symptoms observed.

[0051] In preferred embodiments of methods for screening for diagnosisof inflammatory response-related diseases or disorders involving theinteraction of PYK2 and a natural binding partner, the inflammatoryresponse-related disease or disorder is selected from the groupconsisting of inflammatory bowel diseases and connective tissuediseases. Preferably, the inflammatory bowel diseases are selected fromthe group consisting of ulcerative colitis and Crohn's Disease and theconnective tissue diseases are selected from the group consisting ofrheumatoid arthritis, systemic lupus erythematosus, progressive systemicsclerosis, mixed connective tissue disease, and Sjögren's syndrome.

[0052] A third aspect of the invention features a method for treating orpreventing a disease or disorder, wherein said disease or disorder ischaracterized by an inflammatory response involving an abnormality in asignal transduction pathway that includes an interaction between a PYK2polypeptide and a natural binding partner, comprising administering to apatient in need of such treatment one or more compounds preferably in apharmaceutically acceptable composition, wherein said one or morecompounds modulate said interaction.

[0053] In preferred embodiments of methods for treating or preventinginflammatory response-related diseases or disorders involving theinteraction of PYK2 and a natural binding partner, the inflammatoryresponse-related disease or disorder is selected from the groupconsisting of inflammatory bowel diseases and connective tissuediseases. Preferably, the inflammatory bowel diseases are selected fromthe group consisting of ulcerative colitis and Crohn's Disease and theconnective tissue diseases are selected from the group consisting ofrheumatoid arthritis, systemic lupus erythematosus, progressive systemicsclerosis, mixed connective tissue disease, and Sjögren's syndrome.

[0054] In other preferred embodiments of the methods for treating orpreventing inflammatory response-related diseases or disorders involvingthe interaction of PYK2 and a natural binding partner, the one or morecompounds modulate (inhibit or promote) the interaction in vitro and/orin vivo. In some preferred embodiments, the interaction is selected fromthe group consisting of PYK2 phosphorylation, PYK2 natural bindingpartner phosphorylation, PYK2 de-phosphorylation, PYK2 natural bindingpartner de-phosphorylation, and complex formation between PYK2 and anatural binding partner.

[0055] In yet other preferred embodiments of methods for treating orpreventing inflammation-related diseases or disorders involving theinteraction of PYK2 and a natural binding partner, the one or morecompounds is selected from the group consisting of tyrphostins,quinazolines, quinoxolines, quinolines, and indolinones.

[0056] In preferred embodiments the agent is therapeutically effectiveand preferably has an EC₅₀, or IC₅₀ of less than or equal to 100 μM,even more preferably less than or equal to 50 μM, and most preferablyless than or equal to 10 μM. Such lower EC₅₀'s or IC₅₀'s areadvantageous since they allow lower concentrations of molecules to beused in vivo or in vitro for therapy or diagnosis. The discovery ofmolecules with such low EC₅₀'s and IC₅₀'s enables the design andsynthesis of additional molecules having similar potency andeffectiveness. Generally, a therapeutically effective amount is betweenabout 1 nmol and 1 μmol of the molecule, depending on its EC₅₀ or IC₅₀,and on the age and size of the patient, and the disease associated withthe patient.

[0057] The term “preventing” refers to decreasing the probability thatan organism contracts or develops an abnormal condition. Thus, in thesecases a patient would include someone who is thought to be at risk forcontracting an abnormal condition. Persons skilled in the art would beable to identify persons who would be considered at risk fromcontracting an abnormal condition.

[0058] The term “treating” refers to having a therapeutic effect and atleast partially alleviating or abrogating an abnormal condition in theorganism. In this case the patient is already been identified as havingan abnormal condition.

[0059] The term “therapeutic effect” refers to the inhibition oractivation of factors causing or contributing to the abnormal condition.A therapeutic effect relieves to some extent one or more of the symptomsof the abnormal condition. In reference to the treatment of abnormalconditions, a therapeutic effect can refer to one or more of thefollowing: (a) an increase or decrease in the infiltration of cells; (b)inhibition of (i.e., slowing or stopping) or increase in cell movement;(c) relieving to some extent one or more of the symptoms associated withthe abnormal condition; and (d) enhancing or inhibiting the function ofthe affected population of cells. Compounds demonstrating efficacyagainst abnormal conditions can be identified as described herein.

[0060] The term “abnormal condition” refers to a function in the cellsor tissues of an organism that deviates from their normal functions inthat organism. An abnormal condition can relate to cell proliferation,cell differentiation, cell function, or cell survival.

[0061] Abnormal cell infiltration conditions include, but are notlimited to, rheumatoid arthritis and chronic inflammatory bowel disease.

[0062] The term “aberration”, in conjunction with the function of akinase in a signal transduction process, refers to a kinase that isover- or under-expressed in an organism, mutated such that its catalyticactivity is lower or higher than wild-type protein kinase activity,mutated such that it can no longer interact with a natural bindingpartner, is no longer modified by another protein kinase or proteinphosphatase, or no longer interacts with a natural binding partner.

[0063] The term “administering” relates to a method of incorporating acompound into cells or tissues of an organism. The abnormal conditioncan be prevented or treated when the cells or tissues of the organismexist within the organism or outside of the organism. Cells existingoutside the organism can be maintained or grown in cell culture dishes.For cells harbored within the organism, many techniques exist in the artto administer compounds, including (but not limited to) oral, parenteral(e.g. intra-venous, intramuscular, sub-cutaneous, and intra-articular)and aerosol applications. The compounds may also be administered in adepot or sustained release formulation. For cells outside of theorganism, multiple techniques exist in the art to administer thecompounds, including (but not limited to) cell microinjectiontechniques, simple diffusion, and carrier techniques.

[0064] The term “pharmaceutically acceptable” or “pharmaceutical” asused herein refers to solutions or components of the pharmaceuticalcomposition that do not prevent the therapeutic compound from exerting atherapeutic effect and do not cause unacceptable adverse side effects.Examples of pharmaceutically acceptable reagents are provided in TheUnited States Pharmacopeia The National Formulary, United StatesPharmacopeial Convention, Inc., Rockville, Md. 1990 and FDA Inactive.Ingredient Guide 1990, (1996) issued by the Division of Drug InformationResources (both are hereby incorporated by reference herein, includingany drawings). Unacceptable side effects vary for different diseases.Generally, the more severe the disease the more toxic effects which willbe tolerated. Unacceptable side effects for different diseases are knownin the art.

[0065] The term “physiologically acceptable” defines a carrier ordiluent that does not cause significant irritation to an organism andpreferably does not abrogate the biological activity and properties ofthe compound.

[0066] The term “carrier” defines a chemical compound that facilitatesthe incorporation of a compound into cells or tissues. For exampledimethyl sulfoxide (DMSO) is a commonly utilized carrier as itfacilitates the uptake of many organic compounds into the cells ortissues of an organism.

[0067] The term “diluent” defines chemical compounds diluted in water(or another solvent) that will dissolve the compound of interest as wellas stabilize the biologically active form of the compound. Many saltsdissolved in buffered solutions are utilized as diluents in the art. Onecommonly used buffered solution is phosphate buffered saline because itmimics the salt conditions of human blood. Because buffer salts cancontrol the pH of a solution at low concentrations, a diluent rarelymodifies the biological activity of a compound.

[0068] The term “solvent” as used herein refers to a chemical compoundthat facilitates the solubilization of compounds of the invention.Examples of solvents include, but are not limited to, pharmaceuticallyacceptable alcohols, such as ethanol and benzyl alcohol;polyoxyhydrocarbyl compounds, such as poly(ethylene glycol);pharmaceutically acceptable surfactants such as CREMOPHOR® EL;polyglycolized lipids, such as GELUCIRE® and LABRASOL®; andpharmaceutically acceptable oils, such as miglyol 812.

[0069] The term “pharmaceutically acceptable alcohol” as used hereinrefers to alcohols that are liquids at about room temperature(approximately 20° C.). These include propylene glycol, ethanol,2-(2-ethoxyethoxy)ethanol (TRANSCUTOL®, Gattefosse, Westwood, N.J.07675), benzyl alcohol, and glycerol.

[0070] The term “polyoxyhydrocarbyl compound” as used herein refers to awater soluble carbohydrate such as glucose, sucrose, maltotriose, andthe like; water soluble carbohydrate derivatives such as gluconic acidand mannitol, and oligosaccharides; and water soluble polymers such aspolyvinylpyrrolidone, poly(vinyl alcohol), and in particular, polyetherssuch as other polyoxyalkylenes including poly(ethylene glycol) or otherwater soluble mixed oxyalkylene polymers and the polymeric form ofethylene glycol. Although polyoxyhydrocarbyl compounds preferablycontain more than one carbon, oxygen, and hydrogen atom, some moleculessuch as poly(ethylene imine) are also included.

[0071] A particularly preferred class of solubilizing polyoxyhydrocarbylmoieties comprises poly(ethylene glycol) (PEG) and PEG derivatives, suchas PEG monomethyl ether. Other suitable PEG derivatives includePEG-silicon derived ethers. Many of these polymers are commerciallyavailable in a variety of molecular weights. Others may be convenientlyprepared from commercially available materials, such as by coupling ofamino-PEG moiety to a haloalkyl silyl or silane moiety.

[0072] Suitable PEGs may vary in molecular weight from about 200 g/molto about 20,000 g/mol or more, more preferably 200 g/mol to 5,000 g/mol,even more preferably 250 g/mol to 1,000 g/mol, and most preferably 250g/mol to 500 g/mol. The choice of a particular molecular weight maydepend on the particular compound chosen and its molecular weight anddegree of hydrophobicity, as well as the particular application forwhich the formulation is to be used.

[0073] The term “pharmaceutically acceptable surfactant” as used hereinrefers to a compound that can solubilize compounds of the invention intoaqueous solutions, if necessary. Preferably for parenteral formulations,the surfactant is a non-ionic surfactant. Examples of pharmaceuticallyacceptable surfactants include POLYSORBATE 80 and other polyoxyethylenesorbitan fatty acid esters, glyceryl monooleate, polyvinyl alcohol,ethylene oxide copolymers such as PLURONIC® (a polyether) and TETRONIC®(BASF), polyol moieties, and sorbitan esters. Preferably ethoxylatedcastor oils, such as CREMOPHOR® EL, are used for the formulation of somecompounds.

[0074] The term “ethoxylated castor oil” as used herein refers to castoroil that is modified with at least one oxygen containing moiety. Inparticular the term refers to castor oil comprising at least one ethoxylmoiety.

[0075] Further, the term “pharmaceutically acceptable surfactant” asused herein in reference to oral formulations, includes pharmaceuticallyacceptable non-ionic surfactants (for examplepolyoxyethylene-polypropylene glycol, such as POLOXAMER® 68 (BASF Corp.)or a mono fatty acid ester of polyoxyethylene (20) sorbitan monooleate(TWEEN® 80), polyoxyethylene (20) sorbitan monostearate (TWEEN® 60),polyoxyethylene (20) sorbitan monopalmitate (TWEEN® 40), polyoxyethylene(20) sorbitan monolaurate (TWEEN® 20) and the like); polyoxyethylenecastor oil derivatives (for example,polyoxyethyleneglycerol-triricinoleate or polyoxyl 35 castor oil(CREMOPHOR® EL, BASF Corp.), polyoxyethyleneglycerol oxystearate(CREMOPHOR® RH 40 (polyethyleneglycol 40 hydrogenated castor oil) orCREMOPHOR® RH 60 (polyethyleneglycol 60 hydrogenated castor oil), BASFCorp.) and the like); or a pharmaceutically acceptable anionicsurfactant.

[0076] The term “polyglycolized lipids” as used herein refers tomixtures of monoglycerides, diglycerides, or triglycerides andpolyethyleneglycol monoesters and diesters formed by the partialalcoholysis of vegetable oil using PEG of 200 g/mol to 2,000 g/mol or bythe esterification of fatty acids using PEG 200 g/mol to 2,000 g/mol andglycerols. Preferably these include GELUCIRE® 35/10, GELUCIRE® 44/14,GELUCIRE® 46/07, GELUCIRE® 50/13, GELUCIRE® 53/10, and LABRASOL®.

[0077] The term “pharmaceutically acceptable oils” as used herein refersto oils such as mineral oil or vegetable oil (including safflower oil,peanut oil, and olive oil), fractionated coconut oil, propylene glycolmonolaurate, mixed triglycerides with caprylic acid and capric acid, andthe like. Preferred embodiments of the invention feature mineral oil,vegetable oil, fractionated coconut oil, mixed triglycerides withcaprylic acid, and capric acid. A highly preferred embodiment of theinvention features Miglyol® 812 (available from Huls America, USA).

[0078] In preferred embodiments, the methods described herein involveidentifying a patient in need of treatment. Those skilled in the artwill recognize that various techniques may be used to identify suchpatients.

[0079] A fourth aspect of the invention features a compositioncomprising one or more compounds identified by any of the methods of theinvention described above or herein. Preferably, this composition isuseful for treating or preventing a disease or disorder, where thedisease or disorder is characterized by an inflammatory responseinvolving an abnormality in a signal transduction pathway that includesan interaction between a PYK2 polypeptide and a natural binding partner.

[0080] In preferred embodiments, the inflammatory response-relateddisease or disorder is selected from the group consisting ofinflammatory bowel diseases and connective tissue diseases. Preferably,the inflammatory bowel diseases are selected from the group consistingof ulcerative colitis and Crohn's Disease and the connective tissuediseases are selected from the group consisting of rheumatoid arthritis,systemic lupus erythematosus, progressive systemic sclerosis, mixedconnective tissue disease, and Sjögren's syndrome.

[0081] A fifth aspect of the invention features methods of makingcompounds potentially useful to treat or to prevent a disease ordisorder, where the disease or disorder is characterized by aninflammatory response that is characterized by an abnormality in asignal transduction pathway, and where the signal transduction pathwayincludes an interaction between a PYK2 polypeptide and a natural bindingpartner, comprising assaying one or more potential compounds for thoseable to modulate the interaction as an indication of a useful compoundand synthesizing the identified compounds. References describing methodsof synthesizing the identified compounds are indicated in the DetailedDescription of the Invention.

[0082] In preferred embodiments, the inflammatory response-relateddisease or disorder is selected from the group consisting ofinflammatory bowel diseases and connective tissue diseases. Preferably,the inflammatory bowel diseases are selected from the group consistingof ulcerative colitis and Crohn's Disease and the connective tissuediseases are selected from the group consisting of rheumatoid arthritis,systemic lupus erythematosus, progressive systemic sclerosis, mixedconnective tissue disease, and Sjögren's syndrome.

[0083] A sixth aspect of the invention features kits comprising acomposition comprising one or more compounds identified by any of themethods of the invention described above or herein. Preferably, thiscomposition is useful for treating or preventing a disease or disorder,where the disease or disorder is characterized by an inflammatoryresponse involving an abnormality in a signal transduction pathway thatincludes an interaction between a PYK2 polypeptide and a natural bindingpartner. The kit preferably further comprises instructions for useeither on a label or using other suitable means as discussed herein inthe Detailed Description of the Invention.

[0084] In other preferred embodiments, the kit comprises additionalcontainer means containing one or more of the following: diluents,carriers, and solvents. Such containers include small glass containers,plastic containers, or strips of plastic or paper. Such containers allowthe efficient transfer of contents from one container to another, suchthat the contents are not contaminated and the contents can be added ina quantitative fashion from one compartment to another. The kit mayadditionally comprise means for administering the composition. Oneskilled in the art will readily recognize that the compositions of theinstant invention can be readily incorporated into one of theestablished kit formats that are well-known in the art.

[0085] In preferred embodiments, the composition contained in the kit isuseful for treating or preventing an inflammatory response-relateddisease or disorder selected from the group consisting of inflammatorybowel diseases and connective tissue diseases. Preferably, theinflammatory bowel diseases are selected from the group consisting ofulcerative colitis and Crohn's Disease and the connective tissuediseases are selected from the group consisting of rheumatoid arthritis,systemic lupus erythematosus, progressive systemic sclerosis, mixedconnective tissue disease, and Sjögren's syndrome.

[0086] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof, andfrom the claims.

BRIEF DESCRIPTION OF THE FIGURES

[0087] These figures are provided for illustration only, and are notconsidered necessary to disclose the invention.

[0088]FIGS. 1a, 1 b, and 1 c show the targeted disruption of the pyk2gene in mice. Partial restriction maps of murine pyk2 locus, the regionsused as a targeting vector, and the expected mutant allele are shown inFIG 1 a. Solid boxes indicate exons. The probe was used for Southernblot hybridization analysis of the genomic DNA from the ES cells andtail biopsy analysis. The solid line under the pyk2 locus marks the PYK2kinase domain. Arrows mark the expected Apal fragments of the wild-typeand mutant alleles. FIG. 1b shows Southern blot hybridization analysisof the mouse tail DNA digested with Apal. FIG. 1c shows immunoblotanalysis with anti-PYK2 antibodies of lysates from Thymus(T), Brain (B),and Spleen (S) from wild-type, pyk2+/−, and pyk2−/− mice. FIG. 1d showsan immunoblot analysis of tissue lysates of FAK from wild type orPYK2−/− mice. B, Brain; T, Thymus; S, spleen; H, Heart; K, Kidney; L,Lung.

[0089]FIG. 2 shows RT-PCR (reverse transcriptase-polymerase chainreaction) analysis of cytokine productions from wild-type +/+ andpyk2−/− thioglycollated-elicited peritoneal macrophages stimulated byLPS. Thioglycollate-elicited peritoneal macrophages were incubated withor without LPS (10 μg/mL) for 3 h, 6 h, 12 h, 24 h, and 48 h. Total RNAswere isolated from cells at each time point and after normalization ofthe amount of mRNA in the total RNAs by RT-PCR of actin mRNA, total RNAswere subjected to RT-PCR with specific probes for various cytokines.

[0090]FIG. 3 shows RT-PCR analysis of the cytokine productions fromwild-type +/+ and pyk2−/− splenocytes stimulated by anti-murine CD3antibodies. Splenocytes were incubated with or without anti-CD3 (1μg/mL) for 12 h, 24 h, 48 h and 72 h. Total RNAs were isolated fromcells at each time point and after normalization of the amount of mRNAin the total RNAs by RT-PCR of the actin mRNA, total RNAs were subjectedto RT-PCR with specific probes for various cytokines.

[0091]FIGS. 4a, 4 b, and 4 c demonstrate Carageenen induced cellularinfiltration in murine air pouches. FIG. 4a shows tissues from wild type+/+ and pyk2−/− air pouches that were treated with Carageenen for 10hours after injection. Samples were formalin-fixed andparaffin-embedded. Sections were stained with Hematoxylin and Eosin.FIG. 4b shows a number of cells infiltrating into wild type +/+ andpyk2−/− air pouches 10 hours after injection with Carageenen. FIG. 4cshows a fraction of cells infiltrating into wild-type +/+ and pyk2−/−mice air pouches 10 hours after injection with Carageenen.

[0092]FIGS. 5a and 5 b demonstrate influenza virus-induced inflammation.FIG. 5a shows histologic sections that were made of the lung frominfluenza virus-infected wild-type +/+ and pyk2−/− mice 4 days afterinfection with the virus. Lungs were formalin-fixed andparaffin-embedded. Sections were stained with Hematoxylin and Eosin.FIG. 5b shows the time course of the survival of mice at different dosesof influenza virus.

[0093]FIGS. 6a and 6 b show the tyrosine phosphorylation of PYK2 inmacrophage cell lines in response to LPS (lipopolysaccharide) andtyrosine kinase inhibitors. Cells were either starved overnight in 0.5%serum and stimulated with LPS for variable time intervals (FIG. 6a), orwere pre-treated with either herbimycin A or genestein for 4 hours andthen stimulated with LPS for 30 minutes (FIG. 6b). After stimulation,cells were washed, lysed, and immunoprecipitated with anti-PYK2antibody. Immunoprecipitates (IP) were then analyzed by westernblotting.

[0094]FIGS. 7a, 7 b, 7 c, and 7 d demonstrate that expression of boththe PYK2 wild-type and the dominant negative kinase mutant are induciblein P388D1 cells. The murine macrophage cell line (P388D1) was seriallytransfected with doxycycline (DOX) inducible plasmids. In the firsttransfection, a regulator plasmid was introduced, and stable,drug-resistant clones were established. Two clones were then selectedand transfected with doxycycline-responsive plasmids containing eitherHA-tagged (hemagglutinin), wild-type PYK2 or the kinase dead,dominant-negative PYK2 mutant (also HA-tagged). After drug selection,stable clones containing the wild-type (FIGS. 7b and 7 d) or thedominant-negative mutant (FIGS. 7a and 7 c) were screened for expressionof the appropriate protein in the presence (FIGS. 7a and 7 b) andabsence (FIGS. 7c and 7 d) of doxycyclin by western blotting.

[0095]FIGS. 8a, 8 b, 8 c, and 8 d compare the secretion of TNF-α bywild-type and kinase mutant P388D1 cells in response to activators ofmacrophage function or PYK2 activity. FIGS. 8a and 8 b show the effectof LPS in the presence and absence of DOX on TNF-α secretion from kinasemutant (dominant negative; FIG. 8a) and wild-type (FIG. 8b) cells. FIGS.8c and 8 d show the effect of PMA in the presence and absence of DOX onTNF-α secretion from kinase mutant (FIG. 8c) and wild-type (FIG. 8d)cells. Induction of P388D1 cells in the presence of doxicycline wascarried out for 48 hours followed by stimulation with LPS (1 μg/mL) orPMA (1 μg/mL) for 18 hrs. Secreted TNF-α was measured by ELISA.

[0096]FIGS. 9a, and 9 b show morphological abnormalities and impairedcell migration in PYK2−/− macrophages. FIG. 9a shows PYK2−/− (b, d, f)or wild type macrophages (a, c, e) were plated on tissue culture dishes.Micrographs of unstimulated (a, b) or SDF1a-stimulated macrophages (c,d, e, f). White arrows mark long multidirectional processes and whitebroken arrows mark multidirectional lamellipodia that are seen inPYK2−/− but not in wild type macrophages. FIG. 9b shows micrographs ofSDF-stimulated or unstimulated PYK2−/− or wild type macrophages platedon tissue culture dishes at different time points. White arrows mark theoriginal point of cell movement, large black arrows mark cellcontraction and small black arrows mark lamellipodia.

[0097]FIGS. 10a and 10 b show measurement of contractile capacity oflamellipodia in wild type or PYK2−/− macrophages by laser tweezers. FIG.10a shows plots of bead displacement from leading edge as a function oftime. Fibronectin coated beads were positioned with tweezers on thelamellipodia of PYK2−/− or wild type macrophages of unstimulated (toppanels) or MIP1α-stimulated (bottom panels) cells near the leading edgeat time 0. The trap remained on for approximately 60 sec. as indicatedby shaded area. Two upper plots present beads displacement onnon-stimulated macrophages and two lower plots present displacement onMIP1α-stimulated macrophages. FIG. 10b shows a histogram of ratio (%) ofbeads displaying escape from trapped field by laser tweezer. All thebeads on the lamellipodia of PYK2−/− or wild type macrophages before andafter stimulation by MIP1α were subjected to a restraining force afterinitial bead-cell contact for 60 sec. Ratios of beads which escaped andmoved rearward were scored. Left column shows score of non-stimulatedmacrophages and right column shows score of MIP1α-stimulatedmacrophages.

[0098]FIGS. 11a, 11 b, 11 c, and 11 d show changes in cytoskeletalorganization in PYK2−/− macrophages. PYK2−/− and wild type macrophagewere plated on tissue culture dishes, fixed by 4% paraformaldehyde andstained by fluorescently labeled phalloidin (A and B) or anti-α-tubulinantibodies (D). In FIG. 11a white arrows in

[0099] (b) and (d) indicate long multi-directional processes and brokenwhite arrows in (b) and (d) indicate membrane ruffles in PYK2−/−macrophages. In FIG. 11b, top and side view of F-actin distribution werevisualized with a confocal microscope. Arrowheads indicate reorganizedF-actin in PYK2−/− macrophages. Black arrows reveal the planes of theslices generated in top and side views. In FIG. 11c, PYK2−/− and wildtype macrophages were placed in MIP1α gradient concentration for 60min., fixed and then stained with fluorescently labeled phalloidin. Thetop left part of the field was exposed to the highest concentration ofMIP1α. White arrows mark regions with strong phalloidin labeling. InFIG. 1d, nonstimulated (a, c) or MIP1α-stimulated (b, d) macrophages.White arrowheads in (b) and (d) demonstrate microtubules assembled atcell periphery in PYK2−/− macrophages. White arrow in (d) demonstratesdecreased intensity of MTOC in PYK2−/− macrophages.

[0100]FIGS. 12a, 12 b, 12 c, and 12 d show a comparison of cellsignaling in wild type and PYK2−/− deficient macrophages. For FIG. 12a,PYK2−/− and wild type macrophages were plated on fibronectin coateddishes for 0.5, 1 and 2 hour, then lysed and incubated withGST-RBD(rho-binding domain) bound to gluthatione beads as described inmaterial and methods. The amount of rhoA: GTP complex was determined byimmunoblotting with anti-rhoA antibodies. For FIG. 12b, the rhoinhibitor C3 was microinjected together with fluorescently labeleddextran into wild type macrophages. After three hours incubation, themorphology of microinjected or non-injected cells was compared byNomarsky microscopy (right). The injected macrophages were identified bytheir fluorescence (left). Arrowheads mark microinjected macrophages,arrows mark lamellipodia and broken arrows mark long process. FIG. 12cshows Ca⁺² release in PYK2−/− or wild type macrophages. Macrophagesloaded by fura-2 were stimulated by MIP1α in Ringer's solution with 2 mMcalcium. Changes in fluorescence intensity as a function of time weretraced in the fura-2 loaded cells following 1-0 stimulation with MIP1αor ATP. FIG. 12d shows production of Ins(1,4,5)P₃ in wild type orPYK2−/− macrophages. Wild type or PYK2−/− macrophages were labeled withmyo-[³H]inositol for 24 hours. After MIP1α stimulation the lipidfraction was extracted and analyzed by HPLC. Closed square indicateproduction of inositol (1,4,5) triphosphate in wild type macrophages andopen circle indicate production of inositol (1,4,5) triphosphate inPYK2−/− macrophages. The experiment was done in duplicates and repeatedthree times.

DETAILED DESCRIPTION OF THE INVENTION

[0101] I. PYK2 and Signal Transduction

[0102] PYK-2 is a non-receptor tyrosine kinase that is activated bybinding of ligand to G-coupled protein receptors such as bradykinin,acetylcholine, and CXCR4 or CCR5. PYK2 has a predicted molecular weightof 111 kD and contains five domains: (1) a relatively long N-terminaldomain; (2) a kinase catalytic domain; (3) a proline rich domain; (4)another proline rich domain; and (5) a C-terminal domain.

[0103] The C-terminal domain of PYK2 has 62% similarity to theC-terminal domain of another non-receptor tyrosine kinase, focaladhesion kinase (FAK), which is also activated by G-coupled proteins.The overall similarity between PYK2 and FAK is 52%. However, theexpression of PYK2 does not correspond with the expression of FAK. PYK2exhibits diffuse cytoplasmic localization unlike the preferentiallocalization of Fak in focal adhesion areas. PYK2 is expressed in avariety of cells including neural tissues, hematopoietic cells, sometumor cell lines, and immune-related cells. PYK2 is highly expressed inthe nervous system and in the adult rat brain.

[0104] PYK2 enzymatic activity is positively regulated byphosphorylation on tyrosine and results in response to binding ofbradykinin, TPA, calcium ionophore, carbachol, TPA+ forskolin, andmembrane depolarization. Activated PYK2 is known to phosphorylate andthus suppress the activity of the delayed rectifier-type K+ channel,termed RAK (also called Kvi.2, RBK2, RCK5 and NGKI), that is highlyexpressed in the brain and cardiac atria. In the same system, FAK doesnot phosphorylate RAK. PYK2 activation may provide a rapid and highlylocalized control mechanism for ion channel function and kinaseactivation induced by stimuli that elevate intracellular calcium.

[0105] PYK2 is activated by, and phosphorylated on tyrosine residues inresponse to a variety of extracellular signals that lead to calciuminflux or calcium release from internal stores resulting in elevation ofintracellular Ca²⁺ concentration. Calcium influx leads to the activationof PYK2, tyrosine phosphorylation of Shc, recruitment of Grb2/Sos andactivation of the MAP kinase signaling pathway that relays signals tothe cell nucleus. Overexpression of PYK2 also leads to activation of MAPkinase. PYK2 has also been shown to be activated by peptide hormonesthat bind to G-protein coupled receptors that mediate theirintracellular responses via Gi and Gq type of G-proteins (Lev, et al.(1995) Nature 376:737-745). Thus, PYK2 may provide a link betweenG-protein coupled receptors and calcium influx and the MAP kinasesignaling pathway; a pathway that relays signals from the cell surfaceto regulate transcriptional events in the nucleus.

[0106] Additionally, in certain cells PYK2 is activated by integrinmediated cell adhesion (Astier, et al. (1997) J. Biol. Chem.272:228-232).

[0107] These results reveal a role for PYK2 in activation of the MAPkinase signaling pathway by ion channels, calcium influx and G-proteincoupled receptors and provide a mechanism for signal transductioninduced by these stimuli. Furthermore, tyrosine phosphorylation of Shcin response to membrane depolarization and carbachol treatment wasdependent on the presence of extracellular calcium, indicating thatcalcium-influx plays a role in regulation of Shc phosphorylation bythese stimuli.

[0108] Similarly, PYK2 may modulate the action of ion channels thatmediate their responses via and are sensitive to intracellular calciumconcentration. PYK2 may therefore provide an autoregulatory role for thevery same channel responsible for PYK2 activation.

[0109] The expression pattern of PYK2 and the external stimuli thatactivate this kinase together with its role in the control of MAP kinasesignaling pathway, suggests a potential role for PYK2 in the control ofa broad array of processes.

[0110] Since PYK2 activity is regulated by intracellular calcium level,both the temporal and spatial pattern of PYK2 activation may represent acarbon copy or a replica of the spatial and temporal profile ofintracellular calcium concentration. Calcium concentration inside cellsis highly localized because of a variety of calcium binding proteinsthat provide a strong buffer. Moreover, in excitable cells the level ofcalcium can be regulated by voltage dependent calcium channels thatinduce large and transient increase in intracellular calciumconcentration leading to calcium oscillations and calcium waves. PYK2may provide a mechanism for rapid and highly localized control of ionchannel function, as well as, localized activation of the MAP kinasesignaling pathway.

[0111] II. Knockout Mice Lacking PYK2

[0112] The current invention demonstrates for the first time in an invivo mouse model and a cellular model the link between PYK2 and theinflammatory response. To demonstrate the role of PYK2 in vivo, knockoutmice lacking the pyk2 gene were created using molecular genetictechniques. FIG. 1 shows the altered size of a fragment of the pyk2 genefollowing the knockout procedure along with the lack of PYK2 expressionin mice which are PYK2−/−. Additionally, Northern blot hybridizationanalysis of total RNA isolated from PYK2−/− brain, thymus and spleentissues did not reveal any PYK2 expression.

[0113] PYK2−/− homozygous mice were fertile and did not show grossanatomical abnormalities compared to wild-type litter-mates, includingtissues that normally express high levels of PYK2, such as the brain,thymus, and spleen. Further analysis of lymphoid subpopulations by flowcytometry did not reveal any obvious difference in the distribution ofT-cells, B-cells, macrophage-monocytes, or NK cells in any lymphoidtissue, including spleen, lymph node, thymus, bone marrow, andperitoneal cavity. Serum levels of IgG1, IgG2a, IgG3, IgM and IgA werealso similar in PYK2−/− and wild-type littermates.

[0114] In the influenza model, the inflammatory response of the knockoutmice was compared with the corresponding mice not containing a pyk2deletion following infection with the influenza virus which leads to anoverwhelming pulmonary inflammatory response and eventually death. Itwas found that the pyk2 knockout mice survived significantly longer thancontrol mice expressing the pyk2 gene (48 hours). Histologic examinationof the lungs of the knockout and control mice showed that in the lungsof the pyk2 knockout mice there was a significantly lower infiltrationof inflammatory cells including PMNs (polymorphonuclear leukocytes),macrophages, and T-cells. In vitro experiments showed that macrophagesand splenocytes from pyk2−/− mice produced decreased amounts ofcytokines and chemokines, including IL-1α, IL-1β, IL-6, IL-10, TNF-α,GMCSF, IFN-γ, and MIP1-α. Lower production of cytokines and chemokinessuch as TNF-α and MIP1-α (among others) results in attenuatedrecruitment and decreased activation of macrophages, T-cells and otherhematopoietic and nonhematopoietic cells involved in the inflammatoryresponse. In addition, macrophages from knockout mice had decreasedmotility in vitro. The attenuated cytokine response and migration defectin vitro correlate with the enhanced survival and decreased pulmonarycellular infiltrate in pyk2−/− mice following influenza challenge.

[0115] In the subcutaneous carageenen air pouch model, a subcutaneousair pouch is surgically induced on the hind flank of the mouse. The airpouch is then filled with the immunogen carageenen, a substance thatinduces an inflammatory response. After 10 hours, the number and type ofcells infiltrating the pouch are enumerated. Histologic examinationrevealed a decrease in the number of cells infiltrating the carageenenfilled pouch of pyk2−/− mice compared to controls. In addition there wasa significant decrease in the number of macrophages present at the siteof inflammation.

[0116] To evaluate the role of PYK2 in mature macrophage cells thatexpress wild-type PYK2, kinase inactive PYK2 protein was introduced intoa normal macrophage cell line. Expression of the kinase inactive PYK2should function as a “dominant-negative” inhibitor and abrogate thefunction of the endogenous wild-type PYK2 protein in these macrophages.Expression of the kinase inactive PYK2 protein decreased secretion ofTNF-α in response to LPS, a physiologically relevant inducer of theinflammatory response.

[0117] Defect in Cell Signaling in PYK2−/− Macrophages

[0118] The experiments presented below show that several intracellularsignals that are stimulated by chemokines are impaired in PYK2−/−macrophages. We found that chemokine-induced production of Ins(1,4,5)P₃as well as other inositol phospholipids is impaired. In addition,chemokine-induced Ca⁺² release and MAP kinase activation are alsoimpaired. In certain cell types intracellular release of Ca⁺² leads tostrong activation of PYK2 (Lev, et al. (1995) Nature 376:737-745). Takentogether, these experiments suggest that PYK2 can function both as aCa⁺² sensor as well as an element crucial for the control of Ca⁺²release.

[0119] Impairment in Macrophage Contractility and Locomotion Caused byPYK2 Deficiency

[0120] The migration of cells in culture in response to extracellularstimuli can be divided into a five-step cycle: (i) extension of theleading edge towards to stimulus; (ii) adhesion of the leading edge tothe substrate, (iii) movement of the cytoplasm towards the leading edge,(iv) release from contact sites at the lagging edge and (V) recycling ofmembrane receptor from the lagging edge to the leading edge of the cell(Sheetz, et al. (1999) Biochem. Soc. Symp. 65:233-243). Each step of thecycle requires orderly changes in cytoskeletal structures and focalcontacts, a process that is regulated by a variety of intracellularenzymes including protein tyrosine kinases and protein tyrosinephosphatases (Manes, et al. (1999) Mol. Cell. Biol. 19:3125-3135).

[0121] The experiments described below demonstrate that in macrophagesPYK2 plays an important role in the control of cell migration. In fact,it appears that PYK2−/− macrophages display alterations in most steps ofthe migration cycle. In PYK2−/− macrophages, extension of the leadingedge is delayed, and multiple extensions are generated (steps i and ii).Furthermore, PYK2−/− macrophages inefficiently detach the lagging edgefrom the matrix to allow net movement (steps iv and v). It appears thatthe migration of the cytoplasm is also impaired in the PYK2 deficientmacrophages. Alterations in the migration cycle are particularly evidentafter the initial extension of lamellipodia, and altered cellpolarization, which could be observed as multi directional lamellipodiaand redistribution of F-actin in multiple sites.

[0122] The strength of the traction force in the lamellipodia of wildtype or PYK2−/− macrophages was analyzed by applying the laser tweezermethod (Choquet, et al. (1997) Cell 88:39-48). The ability of plasmamembrane bound beads to move rearward on the cell surface in oppositionto the restraining force imposed by the force field of the laserprovides a measurement for the ability of cells to migrate over a fixedpoint on the matrix. Measurements of bead movements on wild type orPYK2−/− lamellipodia revealed the diminished capacity ofintegrin/cytoskeletal complex in PYK2−/− macrophages to supply thecontractile force necessary for cell migration. This can be caused bydecreased tightness of the linkage between integrin and the cytoskeletonor decreased contractile force of the cytoskeleton or decreases in bothprocesses. It appears that aberrant adhesion and diminished tractionforce in PYK2−/− macrophages will stabilize lamellipodia that wouldnormally be retracted resulting in the disruption of cellularpolarization and migration.

[0123] It is thought that cells subjected to a chemotactic gradient are“sampling” the environment by extending lamellipodia in severaldirections. Stable attachments to the extracellular matrix are formed inthe direction of the highest concentration of the stimulus, whereasother lamellipodia eventually retract into the cell. The maintenance ofan appropriate cellular polarization in a gradient thus requiresregulation of contact stability and contractile capacity of thelamellipodia. In PYK2−/−macrophages both process are impaired.

[0124] Aberrant regulation of the stability of contacts in PYK2−/−macrophages leads to increased adherence of extended lamellipodia to thesubstratum and impairment in subsequent retraction even when thelamellipodia extends for the purpose of “sampling” the environment intoa direction which does not contain a high concentration of chemokine.Increase in the stability of contacts may result in change in cellpolarization.

[0125] PYK2 Deficiency Impairs rho Activation and Causes CytoskeletalChanges

[0126] Cell morphological change such as contraction of lamellipodia orformation of contact require vigorous changes in cytoskeleton. Numerousstudies have indicated that members of the rho family of GTPases play animportant role in modulating the cytoskeleton in response toextracellular stimuli. Rho GTPases were implicated in the controlcytoskeletal organization, actomyosin contraction, vesicle transport,phospholipid production (Exton, et al. (1997) Eur. J. Biochem.243:10-20) as well as in the control of integrin clustering (Hotchin andHall (1995) J. Cell. Biol. 131:1857-1865; Renshaw, et al. (1996) J.Biol. Chem. 271:21691-21694). In this report we show that activation ofrho in response to integrin-mediated cell adhesion on fibronectin isimpaired in PYK2−/− macrophages. Moreover, formation of long processesand decreased cell contraction displayed by PYK2−/− macrophages alsooccur in wild type macrophages that were treated with a specific rhoinhibitor. Since activation of rho has been shown to be critical forcontraction of lamellipodia (Kimura, et al. (1996) Science 273:245-248),the decreased contractility in PYK2−/− macrophages could be due todecreased activity of rho.

[0127] Degradation of molecules and disassembly of F-actin at the rearend of migrating macrophages are required for cell detachment from thesubstratum. Intracellular calcium plays an important role in thisprocess by regulating the activity of calpain and gelsolin (Witke, etal. (1995) Cell 81:41-51; Huttenlocher, et al. (1997) J. Biol. Chem.272:32719-32722). The attenuated chemokine-induced calcium releasecaused by PYK2 deficiency may lead to the impairment of detachment ofthe rear end of migrating cell.

[0128] Phagocytotic cells such as macrophages or neutrophils as well aslymphocytes migrate quickly into inflammatory regions in response tochemokines and other cues. Recruited inflammatory cells produce abattery of cytokines and a variety of inflammatory mediators such asoxygen radicals, nitric oxide and lipid mediators, which induceinflammatory reaction and systematic effects. The onset of experimentalallergic encephalitis (EAE) induced by injection of a peptidecorresponding to myelin specific protein requires appropriate migrationof macrophages into primary inflammatory region that was triggered byinfiltrated TH1 cells. Experiments presented in this report demonstratethat the onset of EAE was delayed by approximately two days in PYK2deficient mice. However, although EAE was delayed in PYK2 deficient micethe outcome of the disease was more severe in these mice. It is alsodemonstrated that the infiltration of macrophages into carageeneninduced inflammatory region is strongly inhibited in PYK2−/− mice. Takentogether, both the in vitro and in vivo experiments presented in thisstudy reveal an important regulatory role for PYK2 in normal function ofmacrophages.

[0129] The above data confirm the role for PYK2 in cytokine release andsupport the importance of PYK2 function in inflammation. Theseexperiments indicate that treatments that inhibit the functioning ofPYK2 will be useful to decrease excessive inflammatory responses,whereas treatments to enhance the functioning of PYK2 will be useful toaugment inadequate immune responses.

[0130] III. Identification of Compounds that Modulate PYK2

[0131] The present invention relates, inter alia, to methods ofdetecting compounds that modulate the interaction of PYK2 with itsnatural binding partners. The modulation can encompass either adecrease, or an increase in the interaction between PYK2 and its naturalbinding partners. The compounds thus identified would be useful in theprevention or treatment of immune-related disorders involving the signaltransduction system and in particular interactions among PYK2 and itsnatural binding partners. The compounds may be present within a complexmixture, for example, serum, body fluid, or cell extracts. Once thecompounds are identified, they can be isolated using techniques wellknown in the art.

[0132] The present invention also encompasses a method of treating orpreventing immune-related diseases in a mammal with one or morecompounds, that modulate PYK2:natural binding partner interactions,comprising administering the compounds to a mammal in an amountsufficient to modulate PYK2:natural binding partner interactions.

[0133] In an effort to discover novel treatments for diseases,biomedical researchers and chemists have designed, synthesized, andtested molecules that inhibit the function of protein kinases. Somesmall organic molecules form a class of compounds that modulate thefunction of protein kinases. Examples of molecules that have beenreported to inhibit the function of protein kinases include, but are notlimited to, bis monocyclic, bicyclic or heterocyclic aryl compounds (PCTWO 92/20642, published Nov. 26, 1992 by Maguire, et al.),vinylene-azaindole derivatives (PCT WO 94/14808, published Jul. 7, 1994by Ballinari, et al.), 1-cyclopropyl-4-pyridyl-quinolones (U.S. Pat. No.5,330,992), styryl compounds (U.S. Pat. No. 5,217,999),styryl-substituted pyridyl compounds (U.S. Pat. No. 5,302,606), certainquinazoline derivatives (EP Application No. 0 566 266 A1), selenoindolesand selenides (PCT WO 94/03427, published Feb. 17, 1994 by Denny, etal.), tricyclic polyhydroxylic compounds (PCT WO 92/21660, publishedDec. 10, 1992 by Dow), and benzylphosphonic acid compounds (PCT WO91/15495, published Oct. 17, 1991 by Dow, et al.), all of which arehereby incorporated by reference herein including any figures, drawings,or tables.

[0134] Compounds that can traverse cell membranes and are resistant toacid hydrolysis are potentially advantageous as therapeutics as they canbecome highly bioavailable after being administered orally to patients.However, many of these protein kinase inhibitors only weakly inhibit thefunction of protein kinases. In addition, many inhibit a variety ofprotein kinases and will therefore cause multiple side-effects astherapeutics for diseases.

[0135] Some indolinone compounds, however, form classes of acidresistant and membrane permeable organic molecules. WO 96/22976(published Aug. 1, 1996 by Ballinari, et al.; hereby incorporated byreference herein including any figures, drawings, or tables) describeshydrosoluble indolinone compounds that harbor tetralin, naphthalene,quinoline, and indole substituents fused to the oxindole ring. Thesebicyclic substituents are in turn substituted with polar moietiesincluding hydroxylated alkyl, phosphate, and ether moieties. PCTPublication WO 98/07695, published March 26, 1998 by Tang, et al. (Lyon& Lyon Docket No. 221/187 PCT), PCT Publication WO 96/40116, publishedDec. 19, 1996 by Tang, et al. (Lyon & Lyon Docket No. 223/298), andInternational Patent Publication WO 96/22976, published Aug. 1, 1996 byBallinari, et al., all of which are incorporated herein by reference intheir entirety, including any drawings, figures, or tables describeindolinone chemical libraries of indolinone compounds harboring otherbicyclic moieties as well as monocyclic moieties fused to the oxindolering. They also teach methods of indolinone synthesis, methods oftesting the biological activity of indolinone compounds in cells, andinhibition patterns of indolinone derivatives.

[0136] Other examples of substances capable of modulating kinaseactivity include, but are not limited to, tyrphostins, quinazolines,quinoxolines, and quinolines. The quinazolines, tyrphostins, quinolines,and quinoxolines referred to above include well known compounds such asthose described in the literature. For example, representativepublications describing quinazolines include Barker, et al., EPOPublication No.0 520 722 A1; Jones, et al., U.S. Pat. No. 4,447,608;Kabbe, et al., U.S. Pat. No. 4,757,072; Kaul and Vougioukas, U.S. Pat.No. 5,316,553; Kreighbaum and Corner, U.S. Pat. No. 4,343,940; Pegg andWardleworth, EPO Publication No. 0 562 734 A1; Barker, et al. (1991)Proc. of Am. Assoc. for Cancer Research 32:327; Bertino, J. R. (1979)Cancer Research 3:293-304; Bertino, J. R. (1979) Cancer Research 9(2part 1)293-304; Curtin, et al. (1986) Br. J. Cancer 53:361-368;Fernandes, et al.(1983) Cancer Research 43:1117-1123; Ferris, et al. J.Org. Chem. 44(2), 173-178; Fry, et al. (1994) Science 265:1093-1095;Jackman, et al. (1981) Cancer Research 51:5579-5586; Jones, et al. J.Med. Chem. 29(6), 1114-1118; Lee and Skibo (1987) Biochemistry 26(23),7355-7362; Lemus, et al.(1989) J. Org. Chem. 54:3511-3518; Ley and Seng,(1975) Synthesis 1975:415-522; Maxwell, et al. (1991) Magnetic Resonancein Medicine 17:189-196; Mini, et al. (1985) Cancer Research 45:325-330;Phillips and Castle (1980) J Heterocyclic Chem. 17(19), 1489-1596;Reece, et al. (1977) Cancer Research 47(11), 2996-2999; Sculier, et al.(1986) Cancer Immunol. and Immunother. 23, A65; Sikora, et al. (1984)Cancer Letters 23:289-295; Sikora, et al. (1988) Analytical Biochem.172:344-355; all of which are incorporated herein by reference in theirentirety, including any drawings.

[0137] Quinoxaline is described in Kaul and Vougioukas, U.S. Pat. No.5,316,553, incorporated herein by reference in its entirety, includingany drawings.

[0138] Quinolines are described in Dolle, et al. (1994) J. Med. Chem.37:2627-2629; MaGuire (1994) J. Med. Chem. 37:2129-2131; Burke, et al(1993) J. Med. Chem. 36:425-432; and Burke, et al. (1992) BioOrganicMed. Chem. Letters 2:1771-1774, all of which are incorporated byreference in their entirety, including any drawings.

[0139] Tyrphostins are described in Allen, et al. (1993) Clin. Exp.Immunol. 91:141-156; Anafi, et al. (1993) Blood 82, 12, 3524-3529;Baker, et al. (1992) J. Cell Sci. 102:543-555; Bilder, et al. (1991)Amer. Physiol. Soc. 6363-6143, C721-C730; Brunton, et al. (1992)Proceedings of Amer. Assoc. Cancer Rsch. 33:558; Bryckaert, et al (1992)Experimental Cell Research 199:255-261; Dong, et al. (1993) J LeukocyteBiology 53:53-60; Dong, et al. (1993) J. Immunol. 151(5), 2717-2724;Gazit, et al. (1989) J. Med. Chem. 32:2344-2352; Gazit, et al. (1993) J.Med. Chem. 36:3556-3564; Kaur, et al. (1994) Anti-Cancer Drugs 5:13-222;King, et al. (1991) Biochem. J. 275:413-418; Kuo, et al. (1993) CancerLetters 74:197-202; Levitzki, A. (1992) The FASEB J 6:3275-3282; Lyall,et al. (1989) J. Biol. Chem. 264:14503-14509; Peterson, et al. (1993)The Prostate 22:335-345; Pillemer, et al. (1992) Int. J. Cancer50:80-85; Posner, et al. (1993) Molecular Pharmacology 45:673-683;Rendu, et al. (1992) Biol. Pharmacology 44(5), 881-888; Sauro andThomas, (1993) Life Sciences 53:371-376; Sauro and Thomas (1993) J.Pharm. and Experimental Therapeutics 267(3), 119-1125; Wolbring, et al.(1994) J. Biol. Chem. 269(36), 22470-22472; and Yoneda, et al. (1991)Cancer Research 51:4430-4435; all of which are incorporated herein byreference in their entirety, including any drawings.

[0140] Other compounds that could be used as modulators includeoxindolinones such as those described in U.S. patent application Ser.No. 08/702,232 filed Aug. 23, 1996 and indolinones such as thosedescribed in U.S. Pat. No. 5,792,783 issued Aug. 11, 1998, entitled“3-Heteroaryl-2-Indolinone Compounds for the Treatment of Disease” (Lyon& Lyon Docket No. 223/301, both of which are hereby incorporated hereinby reference in their entirety, including any drawings, figures ortables.

[0141] IV. Pharmaceutical Formulations and Routes of Administration

[0142] The compounds described herein can be administered to a humanpatient per se, or in pharmaceutical compositions where they are mixedwith other active ingredients, as in combination therapy, or suitablecarriers or excipient(s). Techniques for formulation and administrationof the compounds of the instant application may be found in “Remington'sPharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latestedition. Preferred routes include oral, transdermal, and parenteraldelivery.

[0143] a) Routes Of Administration.

[0144] Suitable routes of administration may, for example, includedepot, oral, rectal, transmucosal, or intestinal administration;parenteral delivery, including intramuscular, subcutaneous, intravenous,intramedullary injections, as well as intrathecal, directintraventricular, intraperitoneal, intranasal, or intraocularinjections.

[0145] Alternately, one may administer the compound in a local ratherthan systemic manner, for example, via injection of the compounddirectly into a solid tumor, often in a depot or sustained releaseformulation.

[0146] Furthermore, one may administer the drug in a targeted drugdelivery system, for example, in a liposome coated with tumor-specificantibody. The liposomes will be targeted to and taken up selectively bythe tumor.

[0147] b) Composition/Formulation

[0148] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

[0149] Pharmaceutical compositions for use in accordance with thepresent invention thus may be formulated in conventional manner usingone or more physiologically acceptable carriers comprising excipientsand auxiliaries that facilitate processing of the active compounds intopreparations that can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

[0150] For injection, the agents of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks's solution, Ringer's solution, or physiological saline buffer.For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

[0151] For oral administration, the compounds can be formulated readilyby combining the active compounds with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained by adding a solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are, in particular, fillers such assugars, including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

[0152] Dragee cores are provided with suitable coatings. For thispurpose, concentrated sugar solutions may be used, which may optionallycontain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,polyethylene glycol, and/or titanium dioxide, lacquer solutions, andsuitable organic solvents or solvent mixtures. Dyestuffs or pigments maybe added to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

[0153] Pharmaceutical preparations that can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

[0154] For buccal administration, the compositions may take the form oftablets or lozenges formulated in conventional manner.

[0155] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebuliser, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

[0156] The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

[0157] Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

[0158] Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

[0159] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0160] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

[0161] A pharmaceutical carrier for the hydrophobic compounds of theinvention is a cosolvent system comprising benzyl alcohol, a nonpolarsurfactant, a water-miscible organic polymer, and an aqueous phase. Thecosolvent system may be the VPD co-solvent system. VPD is a solution of3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80,and 65% w/v polyethylene glycol 300, made up to volume in absoluteethanol. The VPD co-solvent system (VPD:D5W) consists of VPD diluted 1:1with a 5% dextrose in water solution. This co-solvent system dissolveshydrophobic compounds well, and itself produces low toxicity uponsystemic administration. Naturally, the proportions of a co-solventsystem may be varied considerably without destroying its solubility andtoxicity characteristics. Furthermore, the identity of the co-solventcomponents may be varied: for example, other low-toxicity nonpolarsurfactants may be used instead of polysorbate 80; the fraction size ofpolyethylene glycol may be varied; other biocompatible polymers mayreplace polyethylene glycol, e.g. polyvinyl pyrrolidone; and othersugars or polysaccharides may substitute for dextrose.

[0162] Alternatively, other delivery systems for hydrophobicpharmaceutical compounds may be employed. Liposomes and emulsions arewell known examples of delivery vehicles or carriers for hydrophobicdrugs. Certain organic solvents such as dimethylsulfoxide also may beemployed, although usually at the cost of greater toxicity.Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various types of sustained-releasematerials have been established and are well known by those skilled inthe art. Sustained-release capsules may, depending on their chemicalnature, release the compounds for a few weeks up to over 100 days.Depending on the chemical nature and the biological stability of thetherapeutic reagent, additional strategies for protein stabilization maybe employed.

[0163] The pharmaceutical compositions also may comprise suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include but are not limited to calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols.

[0164] Many of the PTK modulating compounds of the invention may beprovided as salts with pharmaceutically compatible counterions.Pharmaceutically compatible salts may be formed with many acids,including but not limited to hydrochloric, sulfuric, acetic, lactic,tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueousor other protonic solvents that are the corresponding free base forms.

[0165] c) Effective Dosage.

[0166] Pharmaceutical compositions suitable for use in the presentinvention include compositions where the active ingredients arecontained in an amount effective to achieve its intended purpose. Morespecifically, a therapeutically effective amount means an amount ofcompound effective to prevent, alleviate or ameliorate symptoms ofdisease or prolong the survival of the subject being treated.Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

[0167] For any compound used in the methods of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating concentration range that includes the IC₅₀ asdetermined in cell culture (i.e., the concentration of the test compoundwhich achieves a half-maximal inhibition of the PTK activity). Suchinformation can be used to more accurately determine useful doses inhumans.

[0168] Toxicity and therapeutic efficacy of the compounds describedherein can be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., for determining the LD₅₀ (thedose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio between LD₅₀ and ED₅₀. Compounds whichexhibit high therapeutic indices are preferred. The data obtained fromthese cell culture assays and animal studies can be used in formulatinga range of dosage for use in humans. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al. (1975) The Pharmacological Basis ofTherapeutics Ch. 1 p. 1).

[0169] Dosage amount and interval may be adjusted individually toprovide plasma levels of the active moiety which are sufficient tomaintain the kinase modulating effects, or minimal effectiveconcentration (MEC). The MEC will vary for each compound but can beestimated from in vitro data; e.g., the concentration necessary toachieve 50-90% inhibition of the kinase using the assays describedherein. Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. However, HPLC assays orbioassays can be used to determine plasma concentrations.

[0170] Dosage intervals can also be determined using MEC value.Compounds should be administered using a regimen that maintains plasmalevels above the MEC for 10-90% of the time, preferably between 30-90%and most preferably between 50-90%.

[0171] In cases of local administration or selective uptake, theeffective local concentration of the drug may not be related to plasmaconcentration.

[0172] The amount of composition administered will, of course, bedependent on the subject being treated, on the subject's weight, theseverity of the affliction, the manner of administration and thejudgment of the prescribing physician.

[0173] d) Packaging

[0174] The compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration. The packor dispenser may also be accompanied with a notice associated with thecontainer in a form prescribed by a governmental agency regulating themanufacture, use, or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the compound for human orveterinary administration. Such notice, for example, may be the labelingapproved by the U.S. Food and Drug Administration or other governmentagency for prescription drugs, or the approved product insert.Compositions comprising a compound of the invention formulated in acompatible pharmaceutical carrier may also be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. Suitable conditions indicated on the label may include, forexample, treatment of immune-related diseases including inflammation,and the like.

[0175] V. Target Diseases to be Treated or Diagnosed by Methods of theInvention

[0176] Target diseases to be treated or diagnosed by the methods of theinvention are generally those that have an aberrant inflammatoryresponse. A pathologic inflammatory response may be a continuation of anacute inflammatory response or a prolonged low-grade inflammatoryresponse, and usually causes permanent tissue damage. Macrophage andT-cell recruitment and functions, such as cytokine production, directlycontribute to inflammatory pathogenesis. There are many types ofdiseases and disorders associated with inflammatory responses, bothacute and chronic, all of which are intended to be included underspecific embodiments of the present invention.

[0177] Specific diseases of interest include, but are not limited to thefollowing:

[0178] Inflammatory bowel diseases include ulcerative colitis andCrohn's disease. The majority of cases of ulcerative colitis are mild,being limited to rectosigmoid involvement. However clinicalmanifestations include bloody diarrhea, mucus, fever, abdominal pain,tenesmus, and weight loss. Complications can include toxic megacolon,colonic perforation, and cancer. The level of risk of cancer is relatedto the extent and duration of colitis, and may be preceded by dysplasia.Intractable disease (to drug treatment), toxic megacolon, cancer andsevere dysplasia may require a colectomy.

[0179] Crohn's disease is generally more serious, occurring in any partof the GI tract with transmural inflammation, bowel wall thickening,linear ulcerations, granulomas, fissures and fistulas. Clinicalmanifestations include fever, abdominal pain, diarrhea, fatigue, weightloss, acute ilietis, anorectal fissures, fistulas, and abscesses.Complications may include intestinal obstruction, intestinal fistulas,and intestinal malignancy. Treatments include parenteral nutrition aswell as pharmaceuticals including corticosteroids, immunosuppressiveagents, and metronidazole. Surgery may be required for fixedobstruction, abscesses, persistent symptomatic fistulas, andintractability.

[0180] Connective tissue diseases involve inflammation of the connectivetissue as well as altered patterns of immunoregulation.

[0181] Rheumatoid arthritis is a serious health care problem.Progressive arthritic conditions in humans cause severe pain, loss ofjoint mobility and disfigurement, and an overall reduction in thequality of life. In rheumatoid arthritis, the synovium hyperproliferates(aided by new blood vessels) and invades the cartilage which isdestroyed. Conventional treatment for rheumatoid arthritis includesnon-steroidal anti-inflammatory drugs (NSAIDs). A need exists for aneffective treatment for rheumatoid arthritis that will disrupt diseaseprogression in addition to suppression or amelioration of symptoms.

[0182] Clinical manifestations of systemic lupus erythematosus includefatigue, fever, malaise, weight loss, skin rashes (malar “butterfly”rash), photosensitivity, arthritis, myositis, oral ulcers, vasculitis,alopecia, anemia, neutropenia, thrombocytopenia, lymphadenopathy,splenomegaly, organic brain syndromes, seizures, psychosis, pleuritis,pericarditis, myocarditis, pneumonitis, nephritis, venous or arterialthrombosis, mesenteric vasculitis, and sicca syndrome. There iscurrently no cure treatment is directed at controlling generalizedinflammation. Drugs include salicylates and NSAIDs. New, effective drugsare desperately needed.

[0183] Clinical manifestations of progressive systemic sclerosis includeRaynaud's phenomenon, scleroderma, telangiectasis, calcinosis,esophageal hypomotility, arthralgias and/or arthritis, intestinalhypofunction, pulmonary fibrosis, hypertension, and renal failure. Renalfailure is the leading cause of death. There is no definitive therapy.

[0184] Clinical manifestations of the mixed connective tissue diseaseinclude Raynaud's phenomenon, polyarthritis, sclerodactyly, esophagealdysfunction, pulmonary fibrosis, and inflammatory myopathy. treatment isdirected to controlling the inflammatory process in general.

[0185] Clinical manifestations of Sjögren's syndrome include xerostomiaand keratoconjunctivitis sicca, nephritis, vasculitis, polyneuropathy,interstitial pneumonitis, pseudolymphoma, autoimmune thyroid disease,and congenital cardiac conduction defects in women with SSA antibodies.Treatment includes symptomatic relief of dryness as well as treatmentsassociated with autoimmune phenomena.

[0186] Although the primary target diseases to be treated or diagnosedby the methods of the invention are those that have an aberrantinflammatory response, other diseases that involve alterations inmacrophage or macrophage-like cell function (i.e. osteoclast) are alsointended to be included. Examples of diseases that are not considered tobe classic inflammatory response-mediated diseases include, but are notlimited to, osteoarthritis, osteoperosis, osteopetrosis andatherosclerosis.

[0187] VI. Other Embodiments

[0188] Methods for evaluation of disorders, methods for monitoringchanges in cells, methods of identifying compounds, methods of isolatingcompounds which interact with a PTK, compositions of compounds thatinteract with a PTK, and derivatives of complexes are disclosed indetail with respect PYK-2 in PCT publication WO 96/18738 and U.S. Pat.No. 5,837,815. These publications are hereby incorporated herein byreference in their entirety, including any drawings, tables, or figures.Those skilled in the art will appreciate that such descriptions areapplicable to the present invention and can be easily adapted to it.Those skilled in the art will also appreciate that any modificationsmade to a complex can be manifested in a modification to any of themolecules in that complex. Thus, the invention includes anymodifications to nucleic acid molecules, polypeptides, antibodies, orcompounds in a complex.

[0189] In addition, the WO 96/18738 provides disclosure describing therecombinant DNA techniques pertaining to the present invention, nucleicacid vectors, the nucleic acid elements of these vectors, the types ofcells that can harbor these vectors, methods of delivering these vectorsto cells or tissues, methods of producing and purifying antibodies,methods of constructing hybridomas that produce these antibodies,methods of detecting signaling molecule complexes, methods of detectinginteractions with natural binding partners, antibodies to complexes,disruption of PTK protein complexes, purification and production ofcomplexes, transgenic animals containing nucleic acid vectors encoding aPTK, antisense and ribozyme approaches, and gene therapy techniques.Those skilled in the art will readily appreciate that such descriptionsare applicable to the present invention and can be easily adapted to it.

[0190] Other methods associated with the invention are described in theexamples disclosed herein.

EXAMPLES

[0191] The examples below are non-limiting and are merely representativeof various aspects and features of the procedures used to demonstratethe role of PYK-2 in signal transduction in inflammation.

[0192] Materials and Methods

[0193] Chemicals

[0194] Bradykinin, pertusis toxin, cholera toxin, forskolin, phorbol12-myristate 13-acetate (PMA), calcium ionophore A23187, carbachol,muscarine, atrophine, mecamylamine, and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) were purchased from Sigma.

[0195] Cells and Cell Culture

[0196] PC12-rat pheochromocytoma cells were cultured in Dulbecco'smodified Eagle's medium containing 10% horse serum, 5% fetal bovineserum, 100 μg/mL streptomycin and 100 units of penicillin/mL. NIH3T3,293, GP+E-86 and PA317 cells were grown in Dulbecco's modified Eagle'smedium containing 10% fetal bovine serum, 100 μg/mL streptomycin and 100units of penicillin/mL.

[0197] Antibodies

[0198] Antibodies against PYK2 were raised in rabbits immunized (HTI)either with a GST fusion protein containing residues 362-647 of PYK2 orwith a synthetic peptide corresponding the 15 amino acids at theN-terminus of PYK2. Antisera were checked by immunoprecipitation andimmunoblot analysis. The specificity was confirmed either by reactivityto the related protein Fak or by competition with the antigenic orcontrol peptides. The antibodies were found to be specific to PYK-2;they do not cross react with FAK.

[0199] Transfections and Infections

[0200] For stable expression in PC12 cells, PYK2 was subcloned into theretroviral vector pLXSN (Miller and Rosman, Biotechniques 7:980,(1989)). The construct was used to transfect GP+E-86 cells usinglipofectamine reagent (GIBCO BRL). 48 hours after transfection, viruscontaining supernatants were collected. Pure retrovirus-containingcell-free supernatants were added to PC12 cells in the presence ofpolybrene (8 μg/mL, Aldrich) for 4 hours (MCB 12 491, 1992). After 24hours, infected PC12 cells were split into growing medium containing 350μL/mg G418. G418 resistant colonies were isolated two to three weekslater and the level of expression was determined by Western blotanalysis.

[0201] Stable cell lines of NIH3T3 that overexpress PYK2 wereestablished by cotransfection of PYK2 subcloned into pLSV together withpSV2neo utilizing lipofectamine reagent (GIBCO BRL). Followingtransfection, the cells were grown in Dulbecco's modified Eagle's mediumcontaining 10% fetal bovine serum and 1 mg/mL G418. Transienttransfections into 293 cells were performed by using a calcium phosphatetechnique, standard in the art.

[0202] Constructs

[0203] GST-PYK2 A DNA fragment of λ900 bp corresponding to residues362-647 of PYK2 was amplified by PCR utilizing the followingoligonucleotide primers: 5′- 5′-CGGGATCCTCATCATCCATCCTAGGAAAGA-3′(sense) and 5′-CGGGAATTCGTCGTAGTCCCAGCAGCGGGT-3′. (antisense)

[0204] The PCR product was digested with BamHI and EcORI and subclonedinto pGEX3X (Pharmacia). Expression of the GST-PYK2 fusion protein wasinduced by 1 mM IPTG essentially as described by Smith, et al. (1988)Gene 67:31. The fusion protein was isolated by electroelution fromSDS-PAGE.

[0205] PYK2

[0206] The full length cDNA sequence of PYK2 was subcloned into thefollowing mammalian expression vectors: pLSV; downstream from the SV40early promoter, pLXSN-retroviral vector; downstream from the Mo-MuLVlong terminal repeat; pRK5; downstream from the CMV promoter.

[0207] PYK2-HA

[0208] The influenza virus hemagglutinin peptide (YPYDVPDYAS) was fusedto the C-terminus of PYK2 utilizing the following oligonucleotideprimers in the PCR reaction: 5′-CACAATGTCTTCAAACGCCAC-3′ and5′-GGCTCTAGATCACGATGCGTAGTCAGGGACATCGTATGGGRACTCTGCAGG TGGGTGGGCCAG-3′.The amplified fragment was digested with RsrII and Xba1 and wassubstituted with the corresponding fragment of PYK2. The nucleotidesequence of the final construct was confirmed by DNA sequencing.

[0209] Kinase Negative Mutant/Dominant-Negative

[0210] In order to construct a kinase negative mutant, Lys (457) wassubstituted to Ala by site directed mutagenesis utilizing the‘Transformer Site-Directed Mutagenesis Kit’ (Clontech). Theoligonucleotide sequence was designed to create a new restriction siteof Nrul. The nucleotide sequence of the mutant was confirmed by DNAsequencing. The oligonucleotide sequence that was used for mutagenesiswas: 5′-CAATGTAGCTGTCGCGACCTGCAAGAA-AGAC-3′ (Nrul site-, Lys-AACsubstituted to Ala-GCG).

[0211] Immunoprecipitation and Immunoblot Analysis

[0212] Cells were lysed in lysis buffer containing 50 mMN-2-hydroxyethylpiperazine-N′-2-ethanesulferic acid (HEPES pH 7.5), 150mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 1 mMethyleneglycol-bis (β-aminoethyl ether)-N,N,N′N′-tetraacetic acid(EGTA), 10 μg leupeptin per mL, 10 μg aprotinin per mL, 1 mMphenylmethylsulfonyl fluoride (PMSF), 200 μM sodium orthovanadate and100 mM sodium fluoride. Immunoprecipitations were performed usingprotein A-sepharose (Pharmacia) coupled to specific antibodies.Immunoprecipitates were washed either with HNTG' solution (20 mM HEPESbuffer at pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, 100 mMsodium fluoride, 200 μM sodium orthovanadate) or successively with H′solution (50 mM Tris-HCl pH 8, 500 mM NaCl, 0.1% SDS, 0.2% Triton X-100,100 mM NaF, 200 μM sodium orthovanadate) and L′ solution (10 mM Tris-HClpH 8, 0.1% Triton X-100, 100 mM NaF, 200 μM sodium orthovanadate). Thewashed immunoprecipitates were incubated for 5 min with gel samplebuffer at 100° C. and analyzed by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE). In some experiments, the gel-embeddedproteins were electrophoretically transferred onto nitrocellulose. Theblot was then blocked with TBS (10 mM Tris pH 7.4, 150 mM NaCl) thatcontained 5% low fat milk and 1% ovalbumin. Antisera or purified mAbswere then added in the same solution and incubation was carried out for1 h at 22° C. For detection, the filters were washed three times (5 mineach wash) with TBS/0.05% Tween-20 and reacted for 45 min at roomtemperature with horseradish peroxidase-conjugated protein A. The enzymewas removed by washing as described above, and the filters were reactedfor 1 min with a chemiluminescence reagent (ECL, Amersham) and exposedto an autoradiography film for 1-15 min.

[0213] In Vitro Kinase Assay

[0214] This was carried out on immunoprecipitates in 50 μL HNTG (20 mMHepes pH 7.5, 150 mM NaCl, 20% glycerol, 0.1% Triton X-100) containing10 mM MnCl₂ and 5 μCi or [mN-³²P]ATP for 20 min at 22° C. The sampleswere washed with H′, M′ and, L′ washing solutions, boiled for 5 min insample buffer and separated by SDS-PAGE.

[0215] Construction of Targeting Vector

[0216] A mouse genomic DNA clone corresponding to the N-terminal domainand the kinase domain, was isolated from 129 strain PI phage library(Genom Systems). 4.5 kb BamH1-Acc1 genomic DNA fragment encoding the 5′terminal of the kinase domain was inserted a XhoI site, flanking to aneo expression cassette in the pPNT vector (Soriano, et al. (1991) Cell64:693-702) and 2 kb AccI-SacI genomic DNA fragment was inserted toBamHI site flanking to HSV thymidine kinase expression cassette in thepPNT vector.

[0217] Electroporation of ES Cells and Generation of PYK2−/− Mice

[0218] R1 ES cells were grown on mitomycin C-treated primary embryonicfibroblast that are extracted from day 15 embryos at 37° C. inDulbecco's modified Eagles Medium (DMEM) supplemented with 15% heatinactivated fetal bovine serum (Hyclone), 0.1 mM 2-mercaptoethanol, 1 mMsodium pyruvate, and 10³ U/ml leukemia inhibitory factor. (LIF) (GIBCO).Cells (7×10⁶) were electroporated in 800 μl of phosphate-bufferedsaline(PBS) with 32 μg of NotI linealized targeting vector DNA at 240V,500 mF using Gene Pulser (Bio-Rad) and plated on gelatin coated plasticdishes. After 48 hours, the cells were transferred to growth mediumsupplemented with G418 (150 mg/ml) (GIBCO) and Gancyclovir (2 mg/ml)(Syntex). G418- and Gancyclovir resistant colonies were picked up 10-12days after electroporation. Homologous recombination was screened bySouthern blot hybridization. Four independently mutated ES cells cloneswere used in embryo aggregation experiments for generation of mice.Chimeric mice were crossed to 129Sv/Ev females and germ linetransmission in heterozyous mice was identified in two independent EScell clone derived F1 mice by Southern blot analysis. Heterozygote micewere intercrossed to produce homozygote mice.

[0219] Cell Culture

[0220] 2.0 ml of 3% Brewer thioglycollate (GIBCO) medium was injectedinto peritoneal cavity 4 days prior to cell harvest. The inflammatorycells, comprised of mixture of macrophages and neutrophils, wereharvested from euthanized animals as lavage by PBS. Macrophages wereisolated as an adherent cells after plating harvested cells onto tissuecultured dishes.

[0221] Immunofluorescence Analysis

[0222] Peritoneal exudate cells were incubated on coverslips for 60minutes. The non-adherent cells were washed off and the adherent cellswere treated for 1 hr with MIP1α: or SDF1α (R&D systems) using byZigmond glass slide chamber (Neuroprobe, Cobin John, MD). The cellsbefore and after treatment were fixed with 4% paraformaldehyde,permabilized with 0.1% Triton X and stained with TRITC-conjugatedphalloidin (Sigma) to visualize F-actin distribution. The slides wereanalyzed using the Leica TCS confocal microscope and images collectedusing Leica TCS User software. Images were further processed using NIHImage 1.61 and Adobe Photoshop.

[0223] Migration Assays

[0224] Thioglycollate-induced peritoneal macrophages were plated ontissue culture dishes and after addition of 100 ng/ml MIP1α, image ofmigrating macrophages on the heated plate at 37° C. were captured every10 minutes using CCD camera equipped with Nikon Diaphot InvertedMicroscope and processed using NIH image software.

[0225] Experimental Inflammation in Air Pouch Model

[0226] The air pouch experiments were conducted essentially as describedpreviously (Wisniewski, et al. (1996) J. Immunol. 156:1609-1615).Briefly, air pouches were induced on the back of wild type and PYK2−/−mice by three subcutaneous injections of air every second day. To inducean acute inflammation, 1 ml of a 2% (w/v) carrageenen solution in thesaline was injected directly into the air pouch. After 10 hour, the micewere sacrificed. 2 ml saline was injected into each air pouch, and theexudate was aspirated. Aliquots were diluted with saline and the cellswere counted. Further, air pouch was dissected, fixed and stained byhematoxyline eosine. Three arbitrarily selected fields each containingapproximately 300 cells were distinguished morphologically and countedper each mouse.

[0227] Microinjection

[0228] Cells were injected in Hepes-buffered culture medium at roomtemperature using a Zeiss (Oberkochen, Germany) Axiopvert 35 microscope,an Eppendorf (Hamburg, Germany) micromanipulator 5170, a microinjector5242 and Sutter capillaries (Novato, Calif.). The holding pressure was450 hPa, the injection pressure was 500 hPa and the duration of theinjection was 200 ms. The concentration of microinjected protein was 1-2mg/ml in PBS. After microinjection, cells were incubated in DMEM at 37°C.

[0229] [³H]Inositol Phospholipids and Ca²⁺ Analyses

[0230] Cells grown in 6-well plates were labeled for 24 h in medium-199containing myo-[³H]inositol(20 μCi/ml). The cells were washed once withPBS and pre-incubated for 10 min. in 2 ml of PBS containing 10 mM LiCl(PH 7.4) at 37° C. prior to the addition of chemokines. The cells wereextracted with methanol/1M HCl/chloroform 1:1:1 and analyzed byhigh-performance liquid chromatography (HPLC) as described (Falasca, etal. (1998) EMBO J. 17:414-422). [Ca²⁺ ]i measurements were performed insingle macrophages as described (Falasca, et al. 1995).

[0231] Laser Trapping Experiments.

[0232] For laser trap experiments, beads were prepared as described(Choquet, et al. (1997) Cell 88:39-48; Felsenfeld, et al. (1999) NatureCell Biology 1:200-206). Briefly, carboxylated latex beads (0.91 μm;Polyscience) were derivitized with carbodimide and coated with ovalbumin(Sigma). The ovalbumin was further derivitized with covalently linkedbiotin (Sulpho NHS-LC biotin; Pierce) to permit the binding of avidin(Neutravidin; Molecular Probes) and finally, a recombinant fragment offibronectin (FN type 3 domains 7-10; FNIII7-10; including the bindingsite for the integrin α5β1).

[0233] The optical gradient laser trap was formed using atitanium-sapphire laser (800 nM; Coherent) which was excited with anargon ion laser (5W; Coherent). The 800 nM laser beam was focusedthrough the bottom port of a Zeiss Axiovert100 TV to form a trap thatwas parfocal with the image plane of the microscope. Bead position wasvisualized using a newvicon camera (MTI Dage) processed through aHamamatsu Argus 10 image processor for background subtraction andcontrast stretching. Video data were collected at 30 frames per secondon an SVHS VCR for subsequent analysis. Analysis of bead position wascarried out using single particle tracking routines (Gelles; 1987)implemented in the ISEE image analysis software package (Innovision)running on an O₂ workstation (Silicon Graphics Inc.).

[0234] Beads were placed at ˜0.5 mm from the edge of the lamellipodiausing a 30 mW (20 pN) trap. The laser trap remained activated until thebead had moved >500 nm from the trap center. Beads that moved <500 nm in30 s.

[0235] Analysis of rho Activation

[0236] Measurement of amount of rho-GTP was performed as previouslydescribed (Ren, et al. (1999) EMBO J. 18:578-585). Briefly, macrophagesplated on the fibronectin were washed with ice-cold PBS and lysed inRIPA buffer (50 mM Tris, pH7.2, 1% Triton X-10, 0.5% sodiumdeoxycholate, 0.1% SDS, 150 mM NaCl, 10 mM MgCl₂, 10 mM/ml eachleupeptin and aprotinin, and 1 mM PMSF). Cell lysates were centrifugedat 13,000 g at 4° C. for 10 min and incubated with GST-RBD (20 μg) beadsat 4° C. for 45 min. The beads were washed four times with washingbuffer (Tris buffer containing 1% Triton X-100, 150 mM NaCl, 10 nMMgCl₂, 10 μg/ml each leupeptin and aprotinin, and 0.1 mM PMSF). BoundRho proteins were detected by immunoblotting using a polyclonal antibodyagainst rho-A (Santa Cruz Biotechnology).

[0237] EAE Induction.

[0238] EAE was induced by subcutaneous injection at the base of the tailof 100 μg MOG 35-55 peptide emulsified in complete Freund's Adjuvant andsupplemented with 2 mg/ml heat-killed M. tuberculosis H37RA (Difco).Pertussis toxin (200 ng/dose; Sigma) was injected intravenously at thetime of MOG injection and 40 hours later.

[0239] Clinical signs of EAE were monitored according to Baron, et al.(1993): 1: limp tail; 2: hind leg weakness; 3: total hind leg paralysis;4: front leg weakness; 5: moribund. Lymphocyte cultures: At varioustime-points, single-cell suspensions from draining (periaortic) andmesenteric lymphnodes were prepared separately and seeded at 10⁶cells/ml in 96 well plates and analyzed for proliferation to stimulationwith anti CD3/28 (1 μg/ml) and to 5 μM MOG 35-55 peptide. After 48 hoursin culture, plates were pulsed with 1 μCi[³H] thymidine (NEN) for 6hours, harvested, and read in a betaplate (Wallac). Culture supernatantswere collected at 24, 48, 72 hours and IL-2, IL-4, IL-10 and IFN-γaccumulation was assessed by ELISA using antibody pairs recommended byPhanningen. Data was collected from individual mice (two mice pertime-point).

[0240] CNS-Infiltrating leukocytes were prepared after total bleeding ofthe mice, cerebellum and spinal cord dissection, incubation withcollagenase D (400 U/ml, Boehringer Mannheim) for 45 minutes at 37° C.and centrifugation in 38% Percoll (Pharmacia). The pellet was washedtwice and stained with anti-Mac1, anti-panCD45, anti I.A.^(b),anti-granulocyte (Gr-1), anti-CD3, and anti-B220, antibodies(Pharmingen).

Example I PYK2 Knockout Experiments

[0241] The Pyk2 gene was knocked out of mice by targeted disruption.FIG. 1a shows partial restriction maps of murine Pyk2 locus, the regionsused as a targeting vector, and the expected mutant allele. Solid boxesindicate exons. The probe was used for Southern blot hybridizationanalysis of the genomic DNA from the ES cells and tail biopsy analysis.The solid line under the Pyk2 locus marks the Pyk2 kinase domain. Arrowsmark the expected Apal fragments of the wild-type and mutant alleles.Southern blot hybridization analysis of the mouse tail DNA digested withApal indicates that the Pyk2 knockout was successful (FIG. 1b).Immunoblot analysis (with anti-Pyk2 antibodies) of lysates from Thymus(T), Brain (B), and Spleen (S) from wild-type, Pyk2+/−, and Pyk2−/− micealso demonstrated the absence of Pyk2 polypeptide in knockout mice (FIG.1c).

EXAMPLE II Analysis of Cytokine Production

[0242] RT-PCR (reverse transcriptase-polymerase chain reaction) was usedto analyze cytokine productions from wild-type +/+ and Pyk2−/−thioglycollate-elicited peritoneal macrophages stimulated by LPS.Thioglycollate-elicited peritoneal macrophages were incubated with orwithout LPS (10 μg/mL) for 3 h, 6 h, 12 h, 24 h, and 48 h. Total RNAswere isolated from cells at each time point. After normalization of theamount of mRNA in the total RNAs by RT-PCR of actin r A, total RNAs weresubjected to RT-PCR with specific probes for various cytokines.

[0243] This experiment reveals a long delay of 24 to 48 hours in theonset of production of cytokines in macrophages derived from PYK2−/−mice (FIG. 2).

[0244] RT-PCR analysis was also used to determine the cytokineproductions from wild-type +/+ and Pyk2−/− splenocytes stimulated byanti-murine CD3 antibodies. Splenocytes were incubated with or withoutanti-CD3 (1 μg/mL) for 12 h, 24 h, 48 h and 72 h. Total RNAs wereisolated from cells at each time point. After normalization of theamount of mRNA in the total RNAs by RT-PCR of the actin mRNA, total RNAswere subjected to RT-PCR with specific probes for various cytokines.

[0245] This experiment reveals a delay of approximately 24 hours in theproduction of cytokines in splenocytes derived from PYK2−/− mice (FIG.3).

[0246] Thus, these experiments showed that macrophages and splenocytesfrom pyk2−/− mice produced decreased amounts of cytokines andchemokines, including IL-1α, IL-1β, IL-6, IL-10, TNF-α, GMCSF, IFN-γ,and MIP1-α. Lower production of cytokines and chemokines such as TNF-αand MIP1-α (among others) results in attenuated recruitment anddecreased activation of macrophages, T-cells and other hematopoietic andnonhematopoietic cells involved in the inflammatory response. Inaddition, macrophages from knockout mice had decreased motility invitro.

Example III Carageenen Model of Inflammation

[0247] In the subcutaneous carageenen air pouch model, a subcutaneousair pouch is surgically induced on the hind flank of the mouse. The airpouch is then filled with the immunogen carageenen, a substance thatinduces an inflammatory response.

[0248] Ten hours after carrageenan injection into wild type or PYK2−/−mice, tissue sections of the injected lesion were examinedmicroscopically for the presence of infiltrating macrophages andneutrophils (FIG. 4a). The average number of infiltrating cells in wildtype mice was 4.8×10⁶ per injected area, while in the PYK2−/− mice therewas an average of only 2.8×10⁶ cells per injected area (FIG. 4b).Morphological examination of the infiltrating cells indicated thatmacrophages comprised approximately 70% of the infiltrate in wild-typemice but only 20% of the infiltrate in the PYK2−/− mice, the remainingcells were primarily neutrophils (FIG. 4b). These data show that thefailure of PYK2−/− macrophages to migrate effectively in vitro iscorrelated with a striking deficit in inflammatory infiltration in vivo.

Example IV Influenza Model of Inflammation

[0249] In the influenza model, infection with the influenza virus leadsto an overwhelming pulmonary inflammatory response and eventually death.

[0250] Influenza virus-induced inflammation in mice lungs was studied inlung sections from influenza virus-infected wild-type +/+ and Pyk2−/−mice 4 days after infection with the virus. Lungs were formalin-fixedand paraffin-embedded. Sections were stained with Hematoxylin and Eosin(FIG. 5a). FIG. 5b shows a time course of the survival of mice atdifferent doses of influenza virus.

[0251] Histologic examination of the lungs of the knockout and controlmice showed that in the lungs of the pyk2 knockout mice there was asignificantly lower infiltration of inflammatory cells including PMNs(polymorphonuclear leukocytes), macrophages, and especially T-cells.Thus, on average the Pyk2 knockout mice exhibited decreased pulmonarycellular infiltrate and lived about 48 hours longer than wild-type miceafter challenge with the influenza virus.

[0252] The attenuated cytokine response and migration defect in vitrocorrelate with the enhanced survival and decreased pulmonary cellularinfiltrate in pyk2−/− mice following influenza challenge.

Example V Analysis of Cytokine Production in PYK2 Dominant-NegativeMutants

[0253] To evaluate the role of PYK2 in mature macrophage cells thatexpress wild-type PYK2, kinase inactive PYK2 protein was introduced intoa normal macrophage cell line. Expression of the kinase inactive PYK2functions as a “dominant-negative” inhibitor and abrogates the functionof the endogenous wild-type PYK2 protein in these macrophages.

[0254] By “dominant negative mutant protein” is meant a mutant proteinthat interferes with the normal PYK2 signal transduction pathway. Thedominant negative mutant protein contains the domain of interest (e.g.,a PYK2 polypeptide or a NBP), but has a mutation preventing propersignaling, for example by preventing binding of a second domain from thesame protein. One example of a dominant negative protein is described inMillauer, et al Nature Feb. 10, 1994. Expression of the kinase inactivePYK2 protein decreased secretion of TNF-α in response to LPS, aphysiologically relevant inducer of the inflammatory response.

[0255] Tyrosine phosphorylation of Pyk2 in response to LPS stimulationwas measured in P388D1 cells. Cells were starved overnight in 0.5% serumand stimulated with LPS for variable time intervals (FIG. 6a). Afterstimulation, cells were washed, lysed, and immuno-precipitated withanti-Pyk2 antibody. Immuno-precipitates were then analyzed by westernblotting. Tyrosine phosphorylated bands were detected with theanti-phosphotyrosine antibody 4G10. Maximal phosphorylation was observedafter 30 minutes. Cells that had been pre-treated with either herbimycinA or genestein for 4 hours were stimulated with LPS for 30 minutes andanalyzed as above (FIG. 6b). Tyrosine phosphorylation was diminished bypre-treatment with non-specific inhibitors of tyrosine phosphorylation.

[0256] The murine macrophage cell line (P388D1) was transfected withdoxycycline inducible plasmids in order to study the effect of adominant-negative Pyk2 protein on macrophage function. In the firsttransfection, a regulator plasmid was introduced and stable,drug-resistant clones were established. These were screened by transienttransfection with a doxycycline-responsive, luciferase reporter plasmid.A range of luciferase responses was obtained from different clones. Twoclones were selected (based upon their doxycycline response) andtransfected with response plasmids containing either HA-tagged,wild-type Pyk2 or the kinase dead Pyk2 mutant (also HA tagged). Afterdrug selection, stable clones were screened for expression of theappropriate protein by western blotting (FIG. 7).

[0257] TNF secretion in response to stimulation with either LPS (1μg/mL) or PMA (1 μg/mL) by the doxycycline inducible clones is shown inFIG. 8. Induction in the presence of doxycycline was carried out for 48hours followed by stimulation with LPS or PMA overnight (18 hours).Cells expressing the kinase-dead Pyk2 mutant had a blunted response toeither LPS or PMA compared with cells expressing the wild-type Pyk2.Secreted TNF-α was measured by ELISA. Both LPS and PMA caused secretionof TNF-αc. Secretion of TNF-α was inhibited by herbimycin A andgenestein, which are non-specific inhibitors of tyrosinephosphorylation.

Example VI PYK2−/− Macrophages Exhibit Abnormal Morphology, EnhancedCell Polarization and Impaired Migration

[0258] Macrophages normally express high levels of PYK2 and bearlydetectable levels of FAK (Lipsky, et al. (1998) J. Biol. Chem.273:11709-11713). It is possible that these cells might be moresusceptible to the loss of PYK2. We have first compared the morphologyof peritoneal macrophages from wild type or PYK2−/− mice 30 minutesafter plating these cells on coverslips or on tissue-culture dishes. Themicrograph depicted in FIG. 9 (panel a) shows that after adhesionwild-type macrophages adopt a typical round shape. Treatment of wildtype macrophages with the chemokine SDF1α resulted in rapid induction oflamellipodia and enhancement of cell spreading (FIG. 9a, panel c and e).In contrast, most PYK2−/− macrophages displayed a flattened morphologywith extensive membrane spreading even without chemokine treatment (FIG.9a, panel b). Addition of SDF1α further increased the formation ofpseudopodia as well as the appearance of long processes (FIG. 9a, paneld and f).

[0259] In view of the enhanced spreading with multiple pseudopodia andlong processes as well as the enhanced substrate attachment displayed byPYK2−/−macrophages, we considered that macrophage migration in responseto chemotactic stimulation could be impaired. We treated wild type andPYK2−/− macrophages with SDF1α and observed cell morphology and movementat ten minute intervals following chemokine stimulation (FIG. 9b). Thisexperiment shows that within ten minutes wild-type macrophages becomepolarized, developing lamellipodia, on one side of the cell. At latertime points (>20 minutes), the cell body moves in the directionestablished by the leading edge, detaching from the substrate at thetrailing edge. In contrast, formation of new lamellipodia by PYK2−/−macrophages in response to SDF1α, was delayed as compared to wild-typecells (FIG. 9b). Furthermore, the cell body showed reduced ability tofollow the leading edge and failed to detach from the substratum. Overtime, the PYK2−/− macrophages extended lamellipodia in severaldirections with similar failure to detach from the substrate.Eventually, most PYK2−/− macrophages developed several pseudopodia-likeprocesses with minimal net migration. Overall, PYK2−/− macrophages areable to form a leading edge in response to a chemotactic stimulus,albeit with slower kinetics. However, these cells are unable to move thecell body after the leading edge efficiently, and fail to detach thelagging edge from the substratum.

Example VII Impairment in Contractile Force in PYK2−/− MacrophagesRevealed by Optical Tweezer Analysis

[0260] Microscopic observation of migrating macrophages revealed thatPYK2−/− cells could extend lamellipodia but the cell body failed to flowinto the newly formed leading edge. We suggested that the contractileactivity of the cytoskelton in the lamellipodia was impaired in PYK2−/−macrophages. The contractile force was determined by measuring therearward movement toward the nucleus of beads coated with recombinantfragment of fibronectin (FN type III domains 7-10) on lamellipodia inopposition to an immobilizing force generated by optical tweezers(Choquet, et al. (1997) Cell 88:39-48; Felsenfeld, et al. (1999) NatureCell Biology 1:200-206). The velocity of rearward movement of the beadsin opposition to this force represents a function of (i) the strength ofassociation between the cytoskeleton and the integrins bound to thefibronectin on the bead, and (ii) the strength of traction forcegenerated by the cytoskeleton itself (Choquet, et al. (1997) Cell88:39-48; Sheetz, et al. (1998) Trends Cell Biol. 8:51-54). Immobilizingforce by optical tweezers was applied to the beads on the lamellipodiaof the cells and the movement of the bound beads was monitored.Representative plots of the distance of bead displacement versus time inwild type or PYK2−/− macrophages is presented in FIG. 10a.

[0261] The beads on the lamellipodia from wild type macrophagesexhibited rearward movement and escaped from the force field of thelaser trap. After chemokine stimulation, the velocity of bead movementon the lamellipodia of wild type macrophages was increased and the beadsescaped more quickly. With the immobilizing force exerted by the opticaltweezers, more than 50% of the beads that were attached to thelamellipodia of either stimulated or unstimulated wild type macrophageswere able to escape from the force field of the optical trap. Incontrast, beads bound to the lamellipodia of PYK2−/− macrophages did notexhibit rearward movement in presence or absence of chemokinestimulation (FIG. 10a). No beads that were attached to the lamellipodiaof either stimulated or unstimulated PYK2−/−macrophages were able toescape from the optical trap (FIG. 10b). Overall, rearward movement,e.g. the contractile force generated by the cytoskeleton, is impaired inPYK2−/− macrophages in comparison to the contractile force generated inwild type macrophages.

Example VIII Altered Cytoskeletal Organization in PYK2−/− Macrophages

[0262] We next examined the status of the cytoskeleton in PYK2−/−macrophages. Visualization of cells stained with fluorescent phalloidinrevealed an increase of F-actin in membrane ruffles in PYK2−/− ascompared to wild-type macrophages (FIG. 11a). Analysis of F-actindistribution by confocal microscopy revealed an increase in reorganizedF-actin underneath the ruffles in PYK2−/− macrophages (FIG. 11b). Whenwild-type macrophages were placed in a chemotactic gradient, phalloidinstaining revealed increase in the relative amount of F-actin at the edgeof the cell in the region that is exposed to the greatest concentrationof chemotactic signal. The distribution of F-actin in stimulated PYK2−/−macrophages was different, in these cells F-actin was distributed atmultiple sites along the cell periphery (FIG. 11c, left). In migratingwild-type macrophages F-actin is continuously redistributed towards theleading edge of the cell. This redistribution of F-actin does not occurin PYK2−/− macrophages, probably resulting in the failure of the cellsto become properly oriented in a chemotactic gradient.

[0263] We have also examined the distribution of microtubules in wildtype and PYK2−/− macrophages. It was proposed that microtubules play animportant role in driving actin polymerization and leading-edgelamellipodia protrusion through specific rho-GTPases during cellmigration (Waterman-Storer, et al. (1999) Nature Cell Biol. 1:45-50).The organization of microtubules in wild type or PYK2−/− macrophages wasvisualized by staining permaebilized cells with anti-tubulin antibodies.The experiment depicted in FIG. 1d shows that the microtubules inPYK2−/− macrophages are more assembled than the microtubules in wildtype macrophages. Upon chemokine stimulation, the microtubules of wildtype macrophages radiate from the microtubules organizing center (MTOC)while PYK2−/− macrophages display long microtubules that are assembledat the cell periphery into longitudinal directions with decreasedintensity towards the MTOC (FIG. 1d). The increased assembly ofmicrotubules in the periphery of PYK2−/− macrophages could be linked tothe enhancement in F-actin organization, extensive lamelliopodiaformation in this region leading to altered cell polarization.

Example IX Impairment in rho Activation in PYK2−/− Macrophages

[0264] The rho family of small G-protein has been implicated in thecontrol of cytoskelal organization leading to changes in cell morphologyand cell migration (Ridley, et al. (1999) Biochem. Soc. Symp.65:111-123). It was demonstrated that integrin-induced cell adhesionleads to the activation of rho (Ren, et al. (1999) EMBO J. 18:578-585).The activated GTP bound form of rho binds to effector proteins that areinvolved in the control of cytoskeletal organization and contraction oflamellipodia (Allen, et al. (1997) J Cell Sci. 110:707-720; Maekawa, etal. (1999) Science 285:895-898). We have analyzed activation of rho inmacrophages by applying a “pull-down” assay using a GST fusion proteincontaining the binding site from rhotekin for the GTP bound form of rho(Ren, et al. (1999) EMBO J. 18:578-585). The experiment presented inFIG. 12a shows fibronectininduced activation of rho as a function oftime. In contrast, a similar experiment performed with PYK2−/−macrophages revealed reduced activation of rho in the mutant macrophagesin response to integrin-induced cell adhesion (FIG. 12a).

[0265] We have further examined the role played by rho in the control ofmacrophage morphology by microinjecting into these cells a specificinhibitor of rho designated C3 (Chardin, et al. (1989) EMBO J.8:1087-1092) together with fluorescently labeled dextran as a specificmarker (FIG. 12b. This experiment demonstrates that wild typemacrophages microinjected with C3, showed a rapid and extensive cellspreading with strong ruffling and formation of long processes similarto the morphological changes seen in PYK2−/− macrophages. However,microinjection of C3 into PYK2−/−macrophages did not cause furtherchanges to those seen in untreated PYK2-−macrophages (FIG. 12b). Takentogether, these experiments show that rho is activated upon adhesion ofmacrophages and that reduced activation of rho may be responsible forthe enhanced spreading, ruffling and formation of long processes inPYK2−/−macrophages.

Example IX Reduced Intracellular Calcium Release and Ins(1,4,5)P₃Production in PYK2Macrophages

[0266] Calcium plays an important role in the control of a variety ofintracellular events as well as in the control of cell shape, and cellmovement (Lawson and Maxfield, 1995). We therefore measured cytoplasmiccalcium release in single cells in response to MIP1α stimulation byusing quantitative fluorescence microscopy of Fura-2 loaded cells.Treatment of wild type macrophages attached to cover slips showedmaximum increase in cytoplasmic [Ca⁺²j concentration at approximately300 nM of MIP1α. By contrast, PYK2−/− adherent macrophages did not showan obvious increase in [Ca⁺²] concentration (FIG. 12c). This experimentshows that PYK2 plays an important role in the control of MIP1α-inducedCa⁺² release in adherent macrophages. Defect in calcium release maycontribute towards the failure of the cells to detach at the rear endleading to impairment in cell migration since proteins that regulate thedegradation of focal contact components and disassembly of F-actinrequire calcium for their action (Witke, et al. (1995) Cell 81:41-51;Kulkarni, et al. (1999) J. Biol. Chem. 274:21265-21275).

[0267] A significant proportion of intracellular Ca⁺² released inresponse to extracellular signals is mediated by inositol (1,4,5)triphosphate [Ins(1,4,5)P₃] production (Furuichi, et al (1989) Nature342:32-38). We therefore analyzed the production of Ins(1,4,5)P₃ inthese cells. In this experiment wild type or PYK2−/−macrophages werelabeled with [³H] myo-inositol and then stimulated with MIP1α. Atvarious times after MI1α stimulation the production of Ins(1,4,5)P₃ wasdetermined by HPLC analysis (Falasca, et al. (1998) EMBO J. 17:414-422).Wild type macrophages showed a biphasic production of Ins(1,4,5)P₃ withpeaks at 20 sec. and 2 min. after MIP1α (stimulation. The experimentpresented in FIG. 12d shows that Ins(1,4,5)P₃ production was severelyreduced in PYK2−/− macrophages; the peak at 20 sec post stimulation wasreduced by approximately 50% as compared to Ins(1,4,5,)P₃ production inwild type macrophages and no Ins(1,4,5)P₃ was generated after 2 min. ofMIP1α stimulation (FIG. 12d). We have also detected impairment in theproduction of glycerophosphoinositol, phosphatidylinositol-3-phosphateand phosphatidylinositol-4-phosphate in PYK2−/− macrophages. Thesefindings suggest that PYM deficiency may lead to a more generalimpairment in phosphatidyl inositol metabolism. We have previously shownthat PYK2 forms a complex with a family of phosphatidylinositol transferproteins designated Nirs both in vitro and in living cells (Lev, et al.(1999) Mol. Cell. Biol. 19:2278-2288). The interaction between PYK2 andthe phosphatidylinositol transfer proteins and its absence in PYK2−/−macrophages may beresponsible for the impairment in phosphatidylinositolmetabolism described.

Example X Delayed Onset of Experimental Autoimmune Encephalomyelitis(EAE) and a More Severe Disease in PYK2−/− Mice.

[0268] Experimental Autoimmune Encephalomyelitis (EAE) is aninflammatory demyelinating disease of the central nervous system (CNS)which exhibits a predominantly mononuclear infiltrate, and is widelyused as an animal model of multiple sclerosis (Zamvil and Steinman(1990) Annu. Rev. Immunol. 8:579-621): Since EAE is largely dependent onthe activity of macrophages, we have compared the susceptibility of wildtype or PYK2−/− mice to EAE by immunizing the mice with MyelinOligodendrocyte Glycoprotein (MOG) (Johns, et al. (1995) J. Immunol.154:5536-5541; Mendel, et al. (1995) Eur. J. Immunol. 25:1951-1959) andmonitoring EAE progression. EAE was induced in PYK2−/− and wild typemice (129Sv) and the clinical course of the disease was monitored daily.As shown in FIG. 13a, both wild type and PYK2−/− mice are susceptible toMOG-induced EAE. However, the onset of EAE was delayed by approximatelytwo days in PYK2 deficient mice as compared to the onset of the diseasein wild-type mice. In several experiments PYK2−/− mice showed lowerincidence of EAE; while not more than 75% of the PYK2−/− mice came downwith the disease, virtually all wild-type mice became sick. However, theoutcome of the disease was more severe in PYK2−/− mice, as compared towild-type mice. For example, in the experiment depicted in FIG. 13, 50%of PYK2−/− mice (4 of 8) died whereas only one of 8 wild-type micesuccumbed, the remaining mice showed partial or complete clinicalrecovery. EAE recovery may not be due entirely to a reduction in theproinflammatory stimulus. There is evidenece that cytokines andregulatory cells are actively involved in the clinical improvement(Welch, et al. (1980) J. Immunol. 125:186-189; Karpus, et al. (1991) J.Immunol. 146:1163-1168; Kennedy, et al. (1992) J. Immunol.149:2496-2505); this process appears to be impaired in PYK2−/− mice.

[0269] Next, draining lymphnode and central nervous system samples wereprepared from wild type or PYK2 deficient mice at different times postinduction. We have shown that T cells from wild type and mutant miceproliferate equally well in response to anti-CD3 stimulation at alltime-points. However, the experiment presented in FIG. 13b shows thatthe proliferative response towards MOG was delayed in T cells fromdraining lymphnodes from PYK2−/− mice as compared to T cells fromwild-type mice.

[0270] One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Themolecular complexes and the methods, procedures, treatments, molecules,specific compounds described herein are presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art which are encompassed within the spirit ofthe invention are defined by the scope of the claims.

[0271] It will be readily apparent to one skilled in the art thatvarying substitutions and modifications may be made to the inventiondisclosed herein without departing from the scope and spirit of theinvention.

[0272] All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains.

[0273] The invention illustratively described herein suitably may bepracticed in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein. Thus, forexample, in each instance herein any of the terms “comprising”,“consisting essentially of” and “consisting of” may be replaced witheither of the other two terms. The terms and expressions which have beenemployed are used as terms of description and not of limitation, andthere is no intention that in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed.

[0274] In particular, although some formulations described herein havebeen identified by the excipients added to the formulations, theinvention is meant to also cover the final formulation formed by thecombination of these excipients. Specifically, the invention includesformulations in which one to all of the added excipients undergo areaction during formulation and are no longer present in the finalformulation, or are present in modified forms.

[0275] In addition, where features or aspects of the invention aredescribed in terms of Markush groups, those skilled in the art willrecognize that the invention is also thereby described in terms of anyindividual member or subgroup of members of the Markush group. Forexample, if X is described as selected from the group consisting ofbromine, chlorine, and iodine, claims for X being bromine and claims forX being bromine and chlorine are fully described.

[0276] Other embodiments are within the following claims.

1 6 1 30 DNA Artificial Sequence Description of Artificial SequencePrimer 1 cgggatcctc atcatccatc ctaggaaaga 30 2 30 DNA ArtificialSequence Description of Artificial Sequence Primer 2 cgggaattcgtcgtagtccc agcagcgggt 30 3 10 PRT Influenza virus 3 Tyr Pro Tyr Asp ValPro Asp Tyr Ala Ser 1 5 10 4 21 DNA Artificial Sequence Description ofArtificial Sequence Primer 4 cacaatgtct tcaaacgcca c 21 5 63 DNAArtificial Sequence Description of Artificial Sequence Primer 5ggctctagat cacgatgcgt agtcagggac atcgtatggg ractctgcag gtgggtgggc 60 cag63 6 31 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide 6 caatgtagct gtcgcgacct gcaagaaaga c 31

1. A method for identifying compounds potentially useful to treat or toprevent a disease or disorder, wherein said disease or disorder ischaracterized by an inflammatory response involving an abnormality in asignal transduction pathway that includes an interaction between a PYK2polypeptide and a natural binding partner, comprising assaying one ormore compounds for those able to modulate said interaction as a means toidentify said potentially useful compounds.
 2. The method of claim 1,wherein said disease or disorder characterized by an inflammatoryresponse is selected from the group consisting of inflammatory boweldiseases and connective tissue disease.
 3. The method of claim 1,wherein said one or more compounds modulate said interaction in vitro.4. The method of claim 1, wherein said one or more compounds modulatesaid interaction in vivo.
 5. The method of claim 1, wherein said one ormore compounds is selected from the group consisting of tyrphostins,quinazolines, quinoxolines, quinolines, and indolinones.
 6. The methodof claim 5, wherein one or more compounds is one or more indolinones. 7.The method of claim 1, wherein said interaction is selected from thegroup consisting of PYK2 phosphorylation, PYK2 natural binding partnerphosphorylation, PYK2 de-phosphorylation, PYK2 natural binding partnerde-phosphorylation, and complex formation between PYK2 and a naturalbinding partner.
 8. A method for diagnosis of a disease or disorder,wherein said disease or disorder is characterized by an inflammatoryresponse involving an abnormality in a signal transduction pathway thatincludes an interaction between a PYK2 polypeptide and a natural bindingpartner, comprising detecting a change in said interaction as anindication of said disease or disorder.
 9. The method of claim 8,wherein said disease or disorder characterized by an inflammatoryresponse is selected from the group consisting of inflammatory boweldiseases and connective tissue diseases.
 10. The method of claim 9,wherein said inflammatory bowel diseases are selected from the groupconsisting of ulcerative colitis and Crohn's Disease.
 11. The method ofclaim 9, wherein said connective tissue diseases are selected from thegroup consisting of rheumatoid arthritis, systemic lupus erythematosus,progressive systemic sclerosis, mixed connective tissue disease, andSjögren's syndrome.
 12. The method of claim 8, wherein said interactionis selected from the group consisting of PYK2 phosphorylation, PYK2natural binding partner phosphorylation, PYK2 de-phosphorylation, PYK2natural binding partner de-phosphorylation, and complex formationbetween PYK2 and a natural binding partner.
 13. The method of claim 8,wherein said change is an increase or decrease in said interaction.14-25. (Canceled).